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number 10 february 1985

EDITORIAL STAFF

John E. Cooper, Editor

Alexa C. Williams, Managing Editor

John B. Funderburg, Editor-in-Chief

Board

Alvin L. Braswell, Curator of David S. Lee, Chief Curator

Lower Vertebrates, N.C. of Birds, N.C

State Museum State Museum

John C. Clamp, Associate Curator William M. Palmer, Chief Curator

(Invertebrates), N.C of Lower Vertebrates, N.C

State Museum State Museum

James W. Hardin, Department Rowland M. Shelley, Chief

of Botany, N.C State Curator of Invertebrates, N.C.

University State Museum

Brimleyana, the Journal of the North Carolina State Museum of Natural His-

tory, will appear at irregular intervals in consecutively numbered issues. Con-

tents will emphasize zoology of the southeastern United States, especially North

Carolina and adjacent areas. Geographic coverage will be limited to Alabama,

Delaware, Florida, Georgia, Kentucky, Louisiana, Maryland, Mississippi, North

Carolina, South Carolina, Tennessee, Virginia, and West Virginia.

Subject matter will focus on taxonomy and systematics, ecology, zoo-

geography, evolution, and behavior. Subdiscipline areas will include general

invertebrate zoology, ichthyology, herpetology, ornithology, mammalogy, and

paleontology. Papers will stress the results of original empirical field studies, but

synthesizing reviews and papers of significant historical interest to southeastern

zoology will be included.

Suitability of manuscripts will be determined by the Editor, and where neces-

sary, the Editorial Board. Appropriate specialists will review each manuscript

judged suitable, and final acceptability will be determined by the Editor.

Address manuscripts and all correspondence (except that relating to subscrip-

tions and exchange) to Editor, Brimleyana, N. C. State Museum of Natural

History, P. O. Box 27647, Raleigh, NC 27611.

In citations please use the full name — Brimleyana.

North Carolina State Museum of Natural History

North Carolina Department of Agriculture

James A. Graham, Commissioner

CODN BRIMD 7

ISSN D193-4406

The Necturus lewisi Study:

Introduction, Selected Literature Review,

and Comments on the Hydrologic Units

and Their Faunas

John E. Cooper and Ray E. Ashton, Jr. '

North Carolina State Museum of Natural History,

P. O. Box 27647, Raleigh, North Carolina 27611

ABSTRACT.— Of the three species of Necturus occurring in North

Carolina, only N. lewisi, the Neuse River Waterdog, is endemic to the

state. Described as a subspecies of N. maculosus by C. S. Brimley in

1924, the salamander occurs in the Neuse and Tar rivers and their

tributaries, from the eastern Piedmont Plateau nearly to tidewater in

the Coastal Plain. Because of its endemicity and limited known distri-

bution, N. lewisi became a candidate for pre-listing studies by the

Office of Endangered Species, U.S. Fish and Wildlife Service, and the

N.C. Wildlife Resources Commission. In 1977, using radioisotope tagg-

ing (60Co), the N.C. State Museum conducted a preliminary behav-

ioral study of N. lewisi, and in 1978 began a 3-year contractual study

of the animal's distribution, ecology, and ethology. Most prior studies

were taxonomic, but some provided information on various aspects of

life history, habitat preference, and preliminary conservation status.

The Neuse and Tar-Pamlico hydrologic units support similar faunas,

and contain other endemic species, some of which are considered by

biologists to be at risk.

INTRODUCTION

Three species of Necturus occur in North Carolina: Necturus macu-

losus maculosus (Rafinesque), the Mudpuppy, inhabits several streams

in the Tennessee River basin of the mountains and has a broad distribu-

tion that ranges from southeastern Canada west to Kansas and south to

northern Alabama; Necturus punctatus punctatus (Gibbes), the Dwarf

Waterdog, occurs in streams and rivers of the Coastal Plain and the

eastern edge of the Piedmont Plateau, ranging along the Atlantic sea-

board from southeastern Virginia to central Georgia; and Necturus

lewisi (Brimley), the Neuse River Waterdog, which is endemic to the

Neuse and Tar-Pamlico river basins in both the eastern Piedmont Pla-

teau and the Coastal Plain, occurring nearly to tidewater. The two east-

ern species, TV. punctatus and N. lewisi, are sympatric and possibly syn-

topic in the Fall Line Zone and parts of the upper Coastal Plain.

1 Present address: International Expeditions. Inc., 1776 Independence

Court, Birmingham, Alabama 35216

Brimleyana No. 10:1-12. February 1985.

2 John E. Cooper and Ray E. Ashton, Jr.

Although six decades have passed since C. S. Brimley (1924) de-

scribed N. lewisi (as a subspecies of N. maculosus), little information on

this large aquatic salamander has been published. Several of the few

papers that included discussions of the taxonomy, distribution, and

ecology of the animal contained incomplete or incorrect information.

This can probably be attributed to the relatively small number of speci-

mens, from very few localities, that were available in collections until

around 1970.

Viosca (1937) elevated N. lewisi to full species status, and Brimley

(1944) seems to have been the first to recognize that the salamander was

restricted to the Neuse and Tar drainages. Since both rivers rise and

debouch in North Carolina (see Figs. 1 and 2 in Braswell and Ashton,

this issue), N. lewisi is endemic to the state. Because its endemicity and

limited known distribution could make it vulnerable to pollution and

habitat modification, and because its "population size and trends are

unknown," Stephan (1977:317-318) advised that N. lewisi be designated

a species of Special Concern. The dearth of information on its distribu-

tion and biology made N. lewisi a candidate for review as part of the

endangered species program in North Carolina. In 1977, the North

Carolina Wildlife Resources Commission provided the North Carolina

State Museum with funds from its Carolina Conservationist Program

for a preliminary behavioral study of the species. Among other accom-

plishments, the study established that radioisotope tagging (60 Co) of N.

lewisi was a reliable method for monitoring the salamander in its natu-

ral habitat (see Ashton, this issue). In 1978, the Wildlife Resources

Commission, through a cooperative agreement with the Office of Endan-

gered Species, U.S. Fish and Wildlife Service (under Title 6 of the

Endangered Species Act of 1973), funded a 3-year contract study of N.

lewisi by the museum. Ray E. Ashton, Jr., formulated the contract pro-

posal and served as director of the project, and Alvin L. Braswell coor-

dinated the extensive field studies. Field technicians were Angelo Cap-

parella, Keith Everett, Ernie Flowers, Paul Freed, Roger Mays, Eric

Rawls, and Jerry Reynolds.

The main goals of the N. lewisi project were to gather information

on the distribution, ecology, and behavior of the species, but the study

yielded results that exceeded these objectives. Data were also collected

on other aspects of the animal's biology; some of these results are

reported elsewhere in this issue. Other data were collected on N. puncta-

tus, which occurs with N. lewisi at many localities but has a broader

distribution. These results will be reported at another time. The general

collections made in both the Neuse and Tar rivers were planned to

include other amphibians, reptiles, fishes, and many kinds of benthic

invertebrates (particularly crayfishes; Cooper and Cooper, in ms.),

without compromising the project's primary objectives. As a result of

Necturus lewisi Study: Introduction 3

this broader emphasis we learned not only a great deal about N. lewisi

and its habitat, but also about its associates. Comments concerning

some of these associates are provided later in this paper.

REVIEW OF SOME PREVIOUS STUDIES

Much of the earlier information on N. lewisi resides in unpublished

sources such as the theses of Hecht (1953) and Fedak (1971), or is

dispersed in published and unpublished sources that are not readily

available. The following is a brief chronological review (with annotation

as appropriate) of some of the more pertinent literature and unpub-

lished manuscripts that have appeared since N lewisi was described. See

Braswell and Ashton (this issue) for review of the literature that deals

specifically with distribution and habitat.

C. S. Brimley (1924) described Necturus maculosus from the Neuse

River near Raleigh, basing his description largely on specimens col-

lected in the Raleigh area since 1894. Nearly all of Brimley's specimens

were caught on hook and line by fishermen. A number of specimens,

including the holotype (USNM 73848), were brought to Brimley by

Frank B. Lewis, hence the patronym. Brimley noted that N m. lewisi

was smaller than N. m. maculosus, and had spotted as opposed to

striped juveniles (less than 3.5 inches long).

Bishop (1926) and Cahn and Shumway (1926) described the adults

and postlarvae or juveniles of N. m. lewisi, but the descriptions of the

larvae left a great deal to be desired. In his tentative revision of the

genus Necturus, Viosca (1937) elevated lewisi to species rank, saying (p.

120) "a study of North Carolina specimens has convinced me that Brim-

ley's form lewisi, described as a subspecies of maculosus, merits full

specific rank...." This decision was largely based on the ventral spotting

pattern, which differed from that of both N. maculosus and N beyeri in

size, number, and color of spots, and on comparative numbers of teeth.

Viosca examined 1 1 juveniles and 4 larvae, described the larval pattern

and coloration, and mentioned that, among other features, the dorsum

lacked spots. He further noted that both dorsal and ventral spotting

increase with age, and are well defined at a length of 90 mm, but failed

to indicate whether this was snout-vent length (SVL) or total length

(TL). Viosca (1937) erroneously gave Brimley's field number (CSB 6868)

as the USNM catalogue number of the holotype (USNM 73848).

Brimley (1939) considered N. lewisi a full species, but Bishop (1941)

retained the trinomial combination. Later, however, Bishop (1943)

accepted species rank for N. lewisi, provided the first photograph of an

adult (a female from Little River, Neuse River basin), and gave a

detailed account of pattern, dentition, and coloration. He also described

a male with swollen cloaca, collected by Lewis on 24 March 1920, and

an egg-laden female that Lewis collected on 8 April 1919. These speci-

4 John E. Cooper and Ray E. Ashton, Jr.

mens led Bishop (1947:34) to suggest "an early-spring mating season for

this species, although some males of maculosus , which has a fall mating

season, are known to retain the swollen glands until spring."

Although Schmidt (1953) retained the trinomial, Hecht (1953)

accepted Viosca's (1937) taxonomic change and placed N lewisi and N

beyeri in a Necturus lewisi superspecies group. Both species differed

from their congeners in having non-striped larvae and spotted medium-

sized adults. The N lewisi superspecies was considered intermediate

between the species N. maculosus and N punctatus. Hecht 's series con-

tained only 20 adult N. lewisi, so he could not address ontogenetic

changes in dentition, body proportions, and other features. He did note

a maximum SVL of at least 175 mm, a minimum breeding size between

100 and 105 mm SVL, and a change to adult pattern at 130 mm SVL.

Hecht (1958) opined that the species of Necturus appeared to be cold-

adapted salamanders, active only in the colder seasons and inactive dur-

ing hottest months. He further speculated that maximum and minimum

breeding size may be an adaptation to thermal regimes of the habitat,

concluding (p. 115): "natural selection has resulted in the adaptation of

the southern species to higher temperatures and a higher metabolism by

reduction of the minimum breeding and maximum size of the species."

He considered the lewisi group the most primitive in the genus, with N.

punctatus an early derivative of the proto-lewisi ancestor, and TV. macu-

losus a direct and recent (advanced) descendent of the lewisi group. He

stated that the striped larva of N maculosus is more specialized than

the primitive unstriped larval type of the lewisi and punctatus groups.

(See Ashton and Braswell, 1979, for discussion of the striped post-

hatchling larva of N. lewisi; also see Sessions and Wiley, this issue, for

chromosome evolution in Necturus.)

Blair et al. (1968) included lewisi as a full species. Neill (1963:173),

defending species status for N. alabamensis Viosca, said that N. lewisi

"most resembles, and is probably most nearly allied to, N. beyeri (sensu

Viosca) even though the two inhabit well-separated portions of the

Coastal Plain. A distribution of this kind, in a group as ancient and

conservative as the waterdogs, suggests that lewisi and beyeri had a

common ancestor in the lowlands that bordered the shoreline of the old

Cretaceous Embayment. As the shoreline retreated southward, exposing

what is now the Coastal Plain, the range of the lewisi-beyeri animal was

fragmented." Brode (1970) revised the genus Necturus, using osteologi-

cal criteria to relegate N. lewisi to subspecies status under N. maculosus,

but this arrangement was not very widely employed.

Fedak (1971), in the most thorough life history study of North

Carolina Necturus to that time, provided information on more than 600

Necturus (punctatus and lewisi) from 32 North Carolina localities, all

collected from the fall of 1966 through the summer of 1969. Of these,

Necturus lewisi Study: Introduction 5

230 were from the Neuse and Tar drainages. Fedak's study showed that

sexual maturity in male N. lewisi occurred at 102 mm SVL, and the first

yolked oocytes and thickened and coiled oviducts were found in females

at 100 mm SVL. Age at sexual maturity was given as 5.5 to 6.5 years.

Male testes were swollen in early fall, and the dark, involuted vasa

deferentia were packed with sperm from November through May. The

cloacal glands were swollen during this period, but swelling progres-

sively decreased from late March through May. Sperm were present in

female spermathecae from December through May, the same period in

which the male cloacal glands were most swollen and the vasa deferentia

loaded with sperm. The largest yolked eggs were found in April and

May, and the smallest in May and July. From these findings Fedak

concluded that N. lewisi (and, from other data, TV. punctatus) mate in

winter, and that egg deposition probably occurs in May or early June.

Fedak (1971:97) also commented on relationships, expressing the

opinion that "Necturus lewisi is probably most closely related to upland

populations oi N. maculosus in the Tennessee River." He further hypoth-

esized that N. lewisi, N maculosus, and N. alabamensis were closely

related, and that N. punctatus was most similar to N. beyeri Viosca.

Stephan (1977) provided a general description of N. lewisi, sum-

marized what was known of its distribution and natural history, then

suggested a conservation status of Special Concern. He also noted (p.

318) that, "The Neuse River Waterdog was considered a species of Spe-

cial Concern at the Workshop on Threatened and Endangered Verte-

brates of the Southeast."

Ashton and Braswell (1979), as part of the preliminary phase of the

overall project, found and described the first reported nest and hatch-

ling larvae of N. lewisi. The nest, discovered on 2 July 1978, was under

a flat rock in 1.2 m of water in the middle of the Little River, northeast-

ern Wake County, about 2 m from shore. Thiry-two empty egg cap-

sules, and three with larvae that soon emerged, were attached to the

underside of the rock. An adult male (147.6 mm SVL) tagged with

60cobalt wire was in attendance in a depression in the sand-gravel sub-

strate directly beneath the eggs. Four other larvae were dip-netted

within 5 m of the nest site. These authors reported that, although hatch-

lings of both N. lewisi and N. maculosus are uniform in color and

nearly indistinguishable, the post-hatchling larvae of N. lewisi have

stripes when between 21 and 41 mm SVL. This striped pattern begins to

fade into the pattern described by Viosca (1937) for specimens of "3!/2

inches" (ca. 90 mm). This was the size considered by Brimley to be

larvae, but we now know that individuals of this size are subadults. The

striped pattern of post-hatchling N. lewisi is quite distinct from that of

post-hatchlings of all other species of Necturus. Ashton and Braswell

(1979:18-19) provided the first illustrations of N lewisi hatchlings and

6 John E. Cooper and Ray E. Ashton, Jr.

older larvae, drawn by Renaldo G. Kuhler, scientific illustrator at the

state museum.

Ashton et al. (1980) reported electrophoretic analyses of 17 loci

coding for enzymes in 20 N. lewisi, 8 unspotted N. punctatus from the

Neuse River drainage, 8 spotted N. punctatus from the Lumber-Pee Dee

drainage, and 21 N. maculosus (1 from North Carolina). They con-

cluded (p. 46): "the specific status of N. lewisi is confirmed by electro-

phoretic data as well as by the distinct larvae described by Ashton and

Braswell (1979). Further, N. punctatus appears to have been reproduc-

tively isolated from sympatric N. lewisi and from allopatric N. maculo-

sus for a considerable period of time, and spotted N. punctatus from the

Pee Dee drainage (North and South Carolina) appear on the basis of

electrophoresis to be genetically similar to the unspotted populations of

the Neuse River system."

Color photographs of adult N. lewisi were provided by Behler and

King (1979) and Martof et al. (1980).

THE HYDROLOGIC UNITS

Both the Neuse and Tar river systems head in the eastern Piedmont

Plateau of the state, drain generally southeast through the Coastal

Plain, then debouch at broad, fairly deep, saline estuaries that feed into

Pamlico Sound. Approximately one-third of each river basin lies in the

Piedmont Plateau and Fall Line Zone (which is some 30 to 40 miles

wide), and two-thirds of each basin is within the Coastal Plain. Not

unexpectedly, the characteristics of the upper hydrologic units differ

considerably from those of the lower basins. The Piedmont Plateau

tributaries flow through valleys of various depths between rolling hills. In

the main, their banks are somewhat precipitous, their floodplains com-

paratively narrow, and their waters graphically lotic, with a combina-

tion of pools and rocky or gravelly rapids and riffles. Substrates are

sand-gravel or sand-silt. Bayless and Smith (1962) recorded average

Piedmont stream gradients of from 14 to 19 feet per mile for the Eno,

Flat, and Little rivers, all of them Neuse feeders, and 2 feet per mile for

the mainstem Neuse. The average Piedmont gradient for the Tar was

reported as 2.8 feet per mile (Smith and Bayless 1964).

By contrast, the Coastal Plain tributaries of both rivers flow

through flatter terrain and have broader floodplains. Their slow-moving

waters have a low average gradient (0.6 feet per mile for the Neuse;

Bayless and Smith 1962). The larger Coastal Plain tributaries often have

high banks and bluffs on their south side, and broad flats and swamps

on their north side (Stuckey 1965). The substrates of these streams are

muck, sand, and detritus. The Coastal Plain streams and rivers are

underlain with relatively soft sedimentary bedrock of from Cretaceous

to Recent age. In the Fall Line Zone this gives way to a bedrock con-

Necturus lewisi Study: Introduction 7

glomerate of metamorphic and igneous rocks of unequal hardness and

resistance to erosive degradation. Within the Piedmont the bedrock is

diversified, primarily granite and other crystalline rocks. North of

Raleigh the Neuse River flows northeast for a few miles in softer sedi-

mentary rocks of Triassic age.

Neuse River

The westernmost headwaters of the Neuse River are tributaries of

the Eno and Flat rivers, and Deep Creek, in the Piedmont Plateau of

southern Person and northeastern Orange counties. Deep Creek conflu-

ences with the South Flat River in northern Durham County to form

the Flat River, and the Flat and Eno rivers confluence at the Durham-

Granville county line northeast of Durham to form the main trunk of

the Neuse. Little River, long known as a lewisi site, is a major eastern

tributary that rises in southwestern Franklin County and confluences

with the mainstem Neuse in central Wayne County, southwest of

Goldsboro. Two large western tributaries — Swift and Middle creeks

— head in southern Wake County and join the Neuse in the Coastal

Plain of central Johnston County, just west of Smithfield. An extensive

Coastal Plain tributary — Contentnea Creek — draining over 980

square miles and with many lower-order tributaries, confluences with

the Neuse River at the junction of Pitt, Lenoir, and Craven counties

northeast of Kinston. Paralleling Contentnea Creek to the east is a

second Swift Creek, which rises in Pitt County and confluences with the

Neuse in Craven County, northwest of New Bern. Another large Coast-

al Plain subsystem, draining about 515 square miles, is the Trent River.

It heads in southern Lenoir and western Jones counties, and empties

into the Neuse River Estuary at New Bern. (See Braswell and Ashton,

this issue, for additional comments on the Trent River). All told, the

Neuse River system drains a watershed of around 6,200 square miles.

Among North Carolina rivers the Neuse River basin is third in area

drained, exceeded only by the Cape Fear (ca. 9,200 sq. mi.) and Yadkin

(ca. 7,200 sq. mi.) river basins.

Tar-Pamlico River

The Tar River has its westernmost headwaters in the Piedmont Pla-

teau of eastern Person, southern Granville, and southern Vance coun-

ties. Its northeastern headwaters are small streams in southern Warren

and Halifax counties. Fishing Creek and its tributaries, which drain an

area of about 760 square miles, comprise a major subdrainage to the

north and east of the mainstem Tar River. Fishing Creek confluences

with the main trunk of the Tar in the Coastal Plain of central Edge-

combe County, north of Tarboro. South of the Fishing Creek subdrain-

age is an area of some 350 square miles drained by Sandy and Swift

8 John E. Cooper and Ray E. Ashton, Jr.

creeks; these streams join the Tar River in Edgecombe County, a few

miles west of the Fishing Creek confluence. The largest Coastal Plain

tributary is Tranters Creek, a slow-moving blackwater stream that rises

in southwestern Martin County, flows south, and confluences with the

Tar northwest of Washington, Beaufort County. East of Washington

the Tar River becomes the Pamlico River, which flows southeast into

the Pamlico River Estuary and then enters Pamlico Sound. The Tar-

Pamlico River system drains a watershed of around 3,100 square miles.

COMMENTS ON THE FAUNAS

Throughout their lengths the Neuse and Tar rivers are parallel sys-

tems, and seem to support nearly identical faunas. Bailey (1977:275)

remarked that the Tar must have been a tributary of the Neuse "during

the late Pleistocene, about 18,000 years ago."

Fishes

Bailey (1977:274) noted that "The Neuse River basin has the richest

recorded fish fauna of our watersheds, though it is only third in drain-

age area." He also pointed out that, except for the white sucker, Catos-

tomus commersoni (Lacep"ede), all of the Tar's fish species also are in

the Neuse, but a number of Neuse species may be absent from the Tar.

An ictalurid — Noturus furiosus Jordan and Meek, the Carolina mad-

tom -- is endemic to both river systems. Bailey et al. (1977:279) consid-

ered this fish a species of Special Concern. Cooper and Braswell (1982)

noted: "Based on the very small numbers of specimens taken in recent

years, despite intensive sampling at many localities in both rivers, the

species seems to have experienced a serious decline." They added, "Its

endemicity and apparent rarity make it vunerable to extinction." In

October 1984, Braswell and Cooper discovered two populations of N.

furiosus in the Tar River, one at a site in the Piedmont Plateau and the

other at a site in the Coastal Plain. The fish was common at both sites.

Thus, N. furiosus may be in less trouble in the Tar River basin than it

appears to be in the Neuse. The Office of Endangered Species, USFWS,

is considering the species for national listing, but not until a pre-listing

study has been completed.

Mussels

At least four species of mussels that occur in either or both of the

rivers were considered in jeopardy by Fuller (1977). "Canthyria" sp., the

Tar River Spiny Mussel, is a unique Tar River species about which

Fuller said (p. 158), "little is known of its natural history, including the

identity of any glochidial host or other aspects of its reproduction." He

considered the species to be Endangered in North Carolina. A recent

study by Johnson and Clarke (1983) indicated that the former range of

this mussel, to which they applied the name Elliptio (Canthyria) stein-

Necturus lewisi Study: Introduction 9

stansana, included the Tar River from Nash to Pitt counties. Today it is

known from a 12-mile section of the river in Edgecombe County, with a

total estimated population of 100 to 500, and is being considered by the

U. S. Fish and Wildlife Service for listing as an Endangered species

(Federal Register 49(1 8 1):3641 8-36420; 17 September 1984). Close rela-

tives of the Tar River Spiny Mussel occur in the James River basin of

Virginia and the Altamaha River basin of Georgia.

The Neuse River population of Carunculina pulla (Conrad), the

Savannah Shoremussel, may have been extirpated, and declines in its

populations elsewhere have been noted. Fuller considered it Endangered

in North Carolina. A third mussel considered Endangered by Fuller was

Prolasmidonta heterodon (Lea), the Ancient Floater. Although known

from a number of river systems, including the Neuse and Tar, Fuller

noted (p. 169) that it was "one of the most rare, elusive, and vulnerable

mollusks in the state and the nation." He also recognized "Lampsilis"

ochracea (Say), the Tidewater Mucket, as a mussel of Special Concern.

One known site of occurrence was the Tar River near Pinetops, Edge-

combe County.

Crustaceans

The decapod crustacean fauna of the Neuse and Tar rivers is com-

paratively rich (both in species and biomass), and probably identical,

with both systems housing at least eight crayfish species and a fresh-

water palaemonid shrimp (listed below). The crayfishes include two

endemic species (indicated in the list by asterisk), one of which is an

Orconectes that appears to be undescribed.

Cambaridae

Cambarus (Depressicambarus) latimanus (LeConte)

Cambarus (Depressicambarus) reduncus Hobbs

Cambarus (Lacunicambarus) diogenes diogenes Girard

Cambarus (Puncticambarus) acuminatus Faxon (sensu lato)

Fallicambarus (Creaserinus) uhleri Faxon

* Orconectes sp. A (Cooper and Cooper 1977:199)

Procambarus (Ortmannicus) acutus acutus (Girard)

* Procambarus (Ortmannicus) medialis Hobbs

Palaemonidae

Palaemonetes paludosus (Gibbes)

Cambarus (D.) reduncus is limited to the Piedmont Plateau. Cam-

barus (D.) latimanus, C. (P.) "acuminatus, " and C. (L.) d. diogenes are

abundant throughout both rivers, with diogenes (an active burrower)

the "rarer" of the three. Orconectes sp. A appears to occur throughout

the Tar River basin, from Granville to Pitt counties, but has not yet

10 John E. Cooper and Ray E. Ashton, Jr.

been found west of the Fall Line Zone in the Neuse drainage. Fallicam-

barus (C.) uhleri and Procambarus (O.) medialis are Coastal Plain spe-

cies, but uhleri has been found along the eastern edge of the Fall Line

Zone in Wake and Franklin counties. Although the type-locality of

medialis is a roadside ditch on U.S. 258, 0.6 miles (1 km) south of

Scotland Neck, Halifax County, the type series and one other lot from

near Scotland Neck are the only collections we know of from the Tar

river basin; all our collections of this species are from the Neuse River

basin. Procambarus (O.) a. acutus is primarily a Coastal Plain species in

both systems, but has been found as far west in the Piedmont as the

Eno River and its tributaries in Orange County. One other species, Pro-

cambarus {Ortmannicus) plumimanus Hobbs and Walton, ostensibly

occurs in the lower Neuse River basin. Its type-locality is in the drainage

of Slocum Creek, Craven County, which empties directly into the Neuse

River Estuary. Nevertheless, none of our Neuse collections contained

plumimanus, but we have found it relatively common in the White Oak

River hydrologic unit.

Other Neuse and Tar crustaceans are now under study, and at least

one species of isopod, preyed upon by N. lewisi, may be an undescribed

endemic.

Miscellany

Centrarchid gamefishes are periodically stocked in the Neuse and

Tar rivers. Information on stocking, physicochemical characteristics,

fish faunas, general macroinvertebrates, and elevation profiles of these

rivers was provided by Bay less and Smith (1962) and Smith and Bayless

(1964). Additional physicochemical data, major sources of effluent dis-

charge, biological and chemical pollutants, general macroinvertebrates,

and phytoplankton of the Neuse River and its tributaries, collected at 23

stations from the Flat and Eno rivers to the mouth of Broad Creek in

the Neuse River Estuary below New Bern, were reported by the Div-

ision of Environmental Management, N.C. Department of Natural

Resources and Community Development (DNRCD 1980).

ACKNOWLEDGMENTS.— We are grateful to the N.C. Wildlife

Resources Commission and the Office of Endangered Species, U.S. Fish

and Wildlife Service, for their support of this study. We thank them,

too, for providing funds that helped defray the cost of this issue of

Brimleyana. Stuart Critcher, Wildlife Resources Commission, was espe-

cially helpful. Other funding was provided by the N.C. State Museum of

Natural History, a division of the N.C. Department of Agriculture.

Patricia S. Ashton developed the basic computer methods used in cer-

tain aspects of the study.

Necturus lewisi Study: Introduction 11

LITERATURE CITED

Ashton, Ray E., Jr., and A. L. Braswell. 1979. Nest and larvae of the Neuse

River Waterdog, Necturus lewisi (Brimley) (Amphibia: Proteidae). Brim-

leyana 1:15-22.

, A. L. Braswell and S. I. Guttman. 1980. Electrophoretic analysis of

three species of Necturus (Amphibia: Proteidae), and the taxonomic status

of Necturus lewisi (Brimley). Brimleyana 4:43-46.

Bailey, Joseph R. 1977. Freshwater fishes. The watersheds and critical areas.

Pp. 268-277 in J. E. Cooper, S. S. Robinson and J. B. Funderburg (Eds.).

Endangered and Threatened Plants and Animals of North Carolina. N. C.

State Mus. Nat. Hist., Raleigh. 444 pp. + i-xvi.

, and Committee. 1977. Freshwater fishes. Species list. Pp. 278-280 in

J. E. Cooper, S. S. Robinson and J. B. Funderburg (Eds.). Endangered and

Threatened Plants and Animals of North Carolina. N. C. State Mus. Nat.

Hist., Raleigh. 444 pp. + i-xvi.

Bayless, Jack D., and W. B. Smith. 1962. Survey and classification of the Neuse

River and tributaries, North Carolina. Final report, Federal Aid in Fish

Restoration, Job I-A, Project F-14-R. N. C. Wildl. Resour. Comm.,

Raleigh. 33 pp. + Appendix A (81 pp.) & Appendix B (14 pp.) (Reprinted

1969).

Behler, John L., and F. W. King. 1979. The Audubon Society Field Guide to

the North American Reptiles and Amphibians. Alfred A. Knopf, New

York. 719 pp.

Bishop, Sherman C. 1926. Notes on the habits and development of the mud-

puppy. N. Y. State Mus. Bull. 268. 38 pp.

1971. The Salamanders of New York. N. Y. State Mus. Bull. 324.

365 pp.

1943. Handbook of Salamanders. Comstock Publ. Co., Ithaca, NY.

555 pp.

1947. Handbook of Salamanders. Second edition. Comstock Publ.

Co., Ithaca, NY. 555 pp.

Blair, Frank W., A. P. Blair, P. Brodkorb, F. R. Cagle and G. A. Moore. 1968.

Vertebrates of the United States. Second edition. McGraw-Hill Book Co.,

NY. 616 pp.

Brimley, C. S. 1924. The water dogs {Necturus) of North Carolina. J. Elisha

Mitchell Sci. Soc. ¥0(3-4): 166-168.

1939-1944. The Amphibians and Reptiles of North Carolina. Caro-

lina Biol. Supply Co.

Brode, W. E. 1970. A systematic study of salamanders of the genus Necturus

(Rafinesque). PhD dissert., Univ. Southern Mississippi, Hattiesburg. 146

pp. Diss. Abstr. Int. B Sci. Eng. 30 (11):5288B-5289B.

Cahn, A. R., and W. Shumway. 1926. Color variation in larvae of Necturus

maculosus. Copeia 1926(1 30) :4-8.

Conant, Roger. 1975. A Field Guide to Reptiles and Amphibians of Eastern and

Central North America. Houghton Mifflin Co., Boston. 429 pp. + i-xviii.

Cooper, John E., and A. L. Braswell. 1982. Norturus furiosus Jordan and

Meek, 1889. Carolina madtom. Account Red Data Book, Conservation

12 John E. Cooper and Ray E. Ashton, Jr.

Monitoring Unit, International Union for Conservation of Nature and

Natural Resources, Cambridge, United Kingdom. 3 pp.

, and M. R. Cooper. 1977. A comment on crayfishes. Pp. 198-199 in

J. E. Cooper, S. S. Robinson and J. B. Funderburg (Eds.). Endangered and

Threatened Plants and Animals of North Carolina. N. C. State Mus. Nat.

Hist., Raleigh. 444+ i-xvi.

, and In manuscript. North Carolina crayfishes (Deca-

poda: Cambaridae): Preliminary key, annotated checklist, and distribut-

ions.

DNRCD. 1980. Working papers: Neuse River Investigation 1979. Div. Environ.

Manage., N. C. Dep. Natur. Res. Commun. Devel., Raleigh. 214 pp. +

Appendix A (43 pp.) & Appendix B (1 p.).

Fedak, Michael A. 1971. A comparative study of the life histories of Necturus

lewisi Brimley and Necturus punctatus Gibbes (Caudata: Proteidae) in

North Carolina. Unpubl. Masters thesis, Duke Univ., Durham. 103 pp.

Fuller, Samuel L. H. 1977. Freshwater and terrestrial mollusks. Pp. 143-194 in

J. E. Cooper, S. S. Robinson and J. B. Funderburg (Eds.). Endangered and

Threatened Plants and Animals of North Carolina. N. C. State Mus. Nat.

Hist., Raleigh. 444 + i-xvi.

Hecht, Max K. 1953. A review of the salamander genus Necturus Rafinesque.

PhD dissert., Cornell Univ., Ithaca. 248 pp.

1958. A synopsis of the mudpuppies of eastern North America. Proc.

Staten Island Inst. Arts Sqi. 2/(1): 1-38.

Johnson, R. I., and A. H. Clarke. 1983. A new spiny mussel, Elliptio (Canthy-

ria) steinstansana (Bivalvia: Unionidae), from the Tar River, North Caro-

lina. Occas. Pap. Mollusks Mus. Comp. Zool. Harvard Univ. 4(61):289-298.

Martof, Bernard S., W. M. Palmer, J. R. Bailey, J. R. Harrison III and J.

Dermid. 1980. Amphibians and Reptiles of the Carolinas and Virginia.

Univ. North Carolina Press, Chapel Hill. 264 pp.

Neill, Wilfred T. 1963. Notes on the Alabama waterdog, Necturus alabamensis

Viosca. Herpetologica 79(3): 166-174.

Schmidt, Karl P. 1953. A Checklist of North American Amphibians and Rep-

tiles. Sixth Edition. Am. Soc. Ichthyol. Herpetol. 280 pp.

Smith, William B., and J. Bayless. 1964. Survey and classification of the Tar

River and tributaries, North Carolina. Final report, Federal Aid in Fish

Restoration, Job I-L, Project F-14-R. N. C. Wild. Resour. Comm., Raleigh.

19 pp., 2 tables, 6 figs. + Appendix A (77 pp.).

Stephan, David R. 1977. Necturus lewisi Brimley. Neuse River Waterdog. Pp.

317-318 in J. E. Cooper, S. S. Robinson and J. B. Funderburg (Eds.).

Endangered and Threatened Plants and Animals of North Carolina. N. C.

State Mus. Nat. Hist., Raleigh. 444 pp. + i-xvi.

Stuckey, Jasper L. 1965. North Carolina: Its Geology and Mineral Resources.

Dep. Conserv. Devel., Raleigh. 550 + i-xviii.

Viosca, Percy, Jr. 1937. A tentative revision of the genus Necturus with descrip-

tions of three new species from the southern Gulf drainage area. Copeia

1937(2): 120-138.

Accepted 20 August 1983

Distribution, Ecology, and Feeding Habits

of Necturus lewisi (Brimley)

Alvin L. Braswell and Ray E. Ashton, Jr. '

North Carolina State Museum of Natural History,

P. O. Box 27647, Raleigh, North Carolina 2761 1

ABSTRACT. — The Neuse River Waterdog, Necturus lewisi Brimley,

is a totally aquatic salamander endemic to the Neuse and Tar River

drainages of North Carolina. A distributional survey, conducted from

December 1978 through May 1979 and January through April 1980,

found N. lewisi at 32% of 361 sites surveyed. Animals predominantly

occurred in streams wider than 15 m, deeper than 100 cm, and with a

main channel flow rate greater than 10 cm/ sec, and were found in

streams from the headwaters of both drainages to the vicinity of salt-

water influence near the coast. None were found in lakes or ponds.

Necturus punctatus was sympatric with N. lewisi at 19 sites along the

Fall Line. Capture success was highest at sites where the substrate con-

sisted mostly of clay or hard soil substrate; however, N. lewisi were

captured on all common substrate types. Some areas with potential

effluent problems yielded no specimens. Activity away from cover was

almost strictly nocturnal, and rising water and high turbidity seemed

to stimulate activity. No animals were trapped when minimum stream

temperatures rose above 18° C. Both larvae and adult N. lewisi

appeared to be opportunistic feeders. Larvae ate a variety of small

aquatic arthropods, while adults expanded their diet to include other

aquatic and some terrestrial invertebrates, along with both aquatic and

terrestrial vertebrates. Sympatric N. punctatus had a similar diet

except for the terrestrial components.

The wide distribution of N. lewisi in the Neuse and Tar River

basins argues against Endangered or Threatened status at this time,

but a conservation status of Special Concern may be warranted due to

the animal's need for larger streams with relatively clean, flowing

water.

INTRODUCTION

Yarrow's (1882) listing of Necturus lateralis (- Necturus maculosus)

from Kinston and Tarboro was the first report of any Necturus from the

Neuse and Tar River drainages. Brimley (1896) said Necturus maculatus

{- N. maculosus) was caught by fishermen in the spring in the Raleigh

area. In 1915, Brimley added Chapel Hill to the range of this sala-

mander, but no voucher specimen has been located to support a locality

in the Cape Fear River drainage. Brimley (1920) discussed 21 specimens

of N. maculosus caught on hook and line in the Neuse River from

1 Present address: International Expeditions, Inc., 1776 Independence

Court, Birmingham, Alabama 35216

Brimleyana No. 10:13-35. February 1985. 13

14 Alvin L. Braswell and Ray E. Ashton, Jr.

November to May in the years 1894 to 1920. Four years later he de-

scribed Necturus lewisi as a subspecies of N. maculosus (Brimley 1924),

based on over 40 specimens collected in the Raleigh area between 1894

and 1924. The type specimen (CSB 6868), taken from the Neuse River

near Raleigh on 25 February 1921 by F. B. Lewis, was deposited in the

National Museum of Natural History (USNM 73848). Brimley indicated

specimens had been taken in the Neuse River by dipnetting "in trash,

often in backwaters, but near swift current". Viosca (1937:138), in his

paper elevating N. m. lewisi to species rank, stated "further north in the

Coastal Plain of North Carolina, the southeastern waterdog, lewisi, and

the least waterdog [Necturus punctatus], are known to occur together in

Little River, a tributary of the Neuse River system." (Little River heads

in the Piedmont Plateau of western Franklin County, crosses the Fall

Line Zone in eastern Wake County and western Johnston County, and

continues into the Coastal Plain of Johnston and Wayne counties. The

collecting site, near Wendell, Wake County, is at the western edge of the

Fall Line Zone.) Viosca erroneously concluded that the probable ranges

of N. lewisi and N. punctatus were "Atlantic Coastal Plain, exclusive of

Florida...." Neither Viosca (1937) nor Bishop (1943) added new locality

or habitat information.

Brimley (1944) described collecting sites along the Neuse River

"where the stream was rapid and usually among clusters of dead leaves

or other rubbish caught in the stream by some obstruction close to the

bank or in a similar mass in a backwater eddy". Hecht (1953:26) con-

cluded that N. lewisi was "a salamander of the larger rivers and deeper

waters" of the Neuse and Tar River drainages of North Carolina. He

expressed doubt (p. 27) about a specimen from the Eno River, Durham

County, which Brimley obtained from a fisherman, saying that it was

"the only one located so far above the Fall Line." He added, "An exam-

ination of the locality revealed it to be very different from any other

localities previously recorded for N. lewisi. The stream is rocky, shallow

and parts of it are temporary during part of the year." As reported

herein, both Fedak (1971) and our field team found N. lewisi in the Eno

River, and other upper tributaries of the Neuse, in both Durham and

Orange counties. Fedak's (1971) unpublished thesis listed collections of

N. lewisi from four localities in the Tar River drainage and nine in the

Neuse River drainage. On the basis of this material he stated that the

primary habitat for N. lewisi, especially in the Piedmont Plateau, was

leaf beds amassed behind obstructions or in backwaters. This echoed

Brimley's (1944) observation. Fedak further indicated a singular lack of

success in collecting N. lewisi under rocks, in riffles, or in fast water. He

also reported collecting N. lewisi and N. punctatus together along the

Fall Line, and showed that a specimen in the collection at Duke Univer-

sity (DU A 1997), from the Lumber River of the Pee Dee drainage and

Necturus lewisi Study: Distribution & Ecology 15

identified by Hecht (1958) as N. lewisi, actually was the spotted form of

N. punctatus. He considered a female Necturus (DU A3458), collected

in the Cape Fear River in Pender County, to be an introduced N. macu-

losus. This decision was based on the animal's weight and the number of

eggs it contained, both of which were well outside the known ranges for

size and fecundity in N. lewisi. Martof et al. (1980) said N. lewisi prefer

to stay in leaf beds in quiet water during the winter. All authors who tried

to collect N. lewisi remarked on the difficulty of capturing specimens.

Collections referred to in the above listed works, and other miscellane-

ous collections made prior to this study, brought the number of known

sites for N. lewisi to about 25 or 30.

Published information on the feeding habits of N. lewisi has been

limited to a brief statement of the results of this study by Nickerson and

Ashton (1983). They said "small aquatic invertebrates, some terrestrial

invertebrates, and some fish and salamanders are included in the diet."

(Nickerson and Ashton also presented an account of the Least Brook

Lamprey, Lampetra aepyptera, being eaten by a captive N. lewisi.)

General range maps and descriptions for N. lewisi were provided in

Conant (1975), Behler and King (1979), and Martof et al. (1980).

The apparent scarcity and relatively small range of N. lewisi, com-

bined with the lack of natual history information, provided justification

for an extensive survey of the Neuse and Tar River drainages to deter-

mine the actual distribution, habitat requirements, and habits of the

species.

MATERIALS AND METHODS

A pilot project to test equipment and techniques was conducted

between November 1977 and July 1978 at a site where N. lewisi was

known to be common. The principal survey period for the main study

was from December 1978 through May 1979 for the Neuse River drain-

age, and January 1980 through April 1980 for the Tar River drainage.

Three full-time technicians were employed during each of the survey

periods. The basin under study was divided into three comparatively

equal parts, and a technician assigned responsibility for sampling each.

Since the duration of the surveys was relatively brief, and the area to be

covered was quite extensive, we had to limit the numbers and locations

of sites as well as the amount of time allocated to sampling each site.

Most survey sites were located near state maintained roads that afforded

easy access to the water. However, several remote sites along the lower

Tar River required using a boat to reach trapping sites.

Commercially available wire minnow traps and Ward's D-frame

dipnets were the principal collecting tools. Seining was productive under

certain conditions, but was done only when two people and appropriate

habitat were available. Set hooks were tried but were discontinued

16 Alvin L. Braswell and Ray E. Ashton, Jr.

because they were less successful and more troublesome to use than

minnow traps. Stream sampling began in the lower, larger sections and

proceeded upstream until N. lewisi could no longer be caught. If N.

lewisi was not taken on the first attempt, a minimum of two trapping

periods (2-3 days each) was alloted to each site. Routinely, 10 minnow

traps were set in a site, and dip nets were often used to sample leaf beds.

Data recorded on a Trap Site Sheet for each site included:

(1) three-digit Site Number;

(2) date sampling began;

(3) county in which site located;

(4) name of stream from USGS topographic maps;

(5) number of nearest state or federal road;

(6) air miles and direction from the center of a nearby town

appearing on a state highway map;

(7) width of stream to the nearest half meter, using a tape or a

Ranging Inc. (model 120) rangefinder;

(8) an average of five evenly spaced measurements across the

stream bed to determine depths (streams too deep to safely

measure by wading were considered over 100cm deep);

(9) flow rate, measured in cm/ sec. at a point of near average

flow for the site (stream width, depth, and average flow rate

were measured at the same point in the stream, and an

attempt was made to take these readings at near normal

water levels);

(10) one of four stream categories based on stream width: little =

5 m or less, small = 5.5 thru 15 m, medium = 15.5 thru 25 m,

large = over 25.5 m (stream categories were established after

sampling was completed and stream width/ capture success

data were examined);

(11) maximum and minimum stream temperatures (°C) occurring

during each sampling period;

(12) temperature change for a period, recorded as no change,

increase, or decrease;

(13) change in water level for a sampling period, recorded to

nearest cm;

(14) water rise or fall for a sampling period, recorded as no

change, rise, or fall;

(15) turbidity measurements, taken with Secchi disc at beginning

and end of each sampling period, and recorded to nearest

cm;

(16) predominant substrate type at each sampling site, listed as

bedrock, loose rocks, sand or gravel, clay or hard soil, or

muck and detritus (including leaf beds);

Necturus lewisi Study: Distribution & Ecology 17

(17) trap days (number of traps x number of days set);

(18) hook days (number of hooks x number of days set);

(19) number of traps set and checked for a sampling period;

(20) number of hooks set and checked for a sampling period;

(21) number of N. lewisi collected during sampling period;

(22) number of N. punctatus collected during sampling period;

and

(23) precipitation during sampling period, to nearest 0.1 cm.

Additional notes on sampling sites included such items as forest

type around sites, channelized vs. natural stream beds, obvious pollu-

tion, and other items deemed significant by the field technician.

All Necturus collected were kept for various biological studies.

Some were shipped alive, frozen, or preserved to other researchers, but

most were killed in a chloretone solution and preserved on the day of

capture. After measurements and weights were taken, the stomachs and

intestines of most juveniles and adults were removed, labeled, and

placed in 75% ethyl alcohol. Stomach contents were later sorted and

identified. Larger Necturus were preserved in 10% formalin. Larval and

small juvenile Necturus were preserved intact in 8% buffered formalin,

and their stomachs removed later. Vertebrate and macroinvertebrate

associates collected in the same habitats as Necturus were also preserved.

The following information was recorded on a Captured Animal

Data sheet for each Necturus specimen:

(1) specimen number, unique to each animal;

(2) site number from Trap Site Sheet;

(3) date specimen collected;

(4) stream name from Trap Site Sheet;

(5) snout-vent length (SVL), measured with mm ruler from tip

of snout to posterior end of cloacal opening on living or

fresh killed specimens;

(6) tail length, measured from posterior end of cloaca to tail tip

on living or fresh killed specimens;

(7) weight of living or fresh killed specimens, recorded to nearest

gram with Ohaus triple beam balance or Presola handheld

scale;

(8) sex, determined by secondary sexual characteristics and dis-

section;

(9) developmental stage, determined by dissection and listed as

"mature", "immature", or "intermediate";

(10) number of eggs (mature or near mature ova);

(11) digestive tract contents, listed as "present" or "absent";

(12) color pattern as: spotted dorsum and venter; spotted dorsum,

plain venter; faint spots on dorsum, sides darker; virtually

plain dorsally, dark sides; totally dark;

18 Alvin L. Braswell and Ray E. Ashton, Jr.

(13) type of bait in trap or on hook;

(14) caught by trap, hook, or net.

All Trap Site Data and Captured Animal Data were coded and

entered into the North Carolina State Government IBM 370/ 168 main

frame computer for SAS analysis. Frequency tables were constructed

for the various environmental and physical parameters to demonstrate

relative capture success for differing factors. Duncan's Multiple Range

Test was used to ascertain significant differences in capture success for

different bottom types, stream depths, stream widths, flow rates, levels

of precipitation, turbidities, stream categories, maximum and minimum

stream temperatures, rises or falls in temperatures, and rises or falls in

water levels.

Necturus voucher specimens were deposited in the lower vertebrate

collections of the North Carolina State Museum of Natural History

(NCSM), and most major collections in the United States were surveyed

for information on N. lewisi specimens. In addition to NCSM, collec-

tion acronyms used herein are: American Museum of Natural History

(AMNH); Carnegie Museum (CM); Duke University (DU); E. E. Brown

personal collection (EEB); Field Museum of Natural History (FMNH);

Museum of Comparative Zoology (MCZ); Tulane University (TU);

University of Florida/ Florida State Museum (UF/FSM), and National

Museum of Natural History (USNM).

RESULTS

A total of 361 sites was sampled for N. lewisi during about 7

months of field work. Sampling efforts totalled 757, and included

15,893 trap days, 2,009 hook days, and an estimated 350 hours dipnet-

ting. Positive sites for N. lewisi numbered 116, which was 32% of the

surveyed sites. A few positive sites duplicated older records, but most

represented new localities. From these sites 208 specimens (58-173 mm

SVL, mean = 130 mm) were caught in minnow traps, 5 (129-154 mm

SVL, mean = 143 mm) on set hooks, and 82 (21-120 mm SVL, mean -

42 mm) in dipnets. Thirty additional specimens were found dead after a

toxic chemical (NaOH) spilled into the Neuse River on 10 July 1980.

An average of 44 trap days was expended per site. In sites positive

for N. lewisi, one was caught in a minnow trap for every 24.5 trap

days. Occasionally, traps were set in the morning and revisited during

the afternoon of the same day; these diurnal trapping periods produced

no Necturus. Shrimp and chicken liver were the most frequently used

baits in minnow traps, and both seemed to work about equally well.

Necturus often engorged themselves with the bait. Dipnetting was most

productive where debris, mostly leaves, accumulated around obstruc-

Necturus lewisi Study: Distribution & Ecology

19

tions in moderate to swift flowing water, or in eddies and backwaters

near moderate to swift flowing water.

Necturus punctatus was collected at 54 sites and was found to be

sympatric with N. lewisi at 19 sites. No N. punctatus were found in the

mainstream of the Neuse or Tar rivers.

All sites surveyed are shown in Figure 1, along with sites of a few

incidental collections of N. lewisi made during the course of this project.

During the project, N. lewisi was collected at 122 sites. Figure 2 includes

historic records and shows all valid sites where N. lewisi has been col-

lected. Appendix A is a list of voucher supported localities and voucher

specimens. The distribution of N. lewisi in both the Neuse and Tar

River drainages extends from headwater streams in the Piedmont Pla-

teau to the vicinity of saline influence near the coast.

Fig. 1. Sites sampled for Necturus lewisi, 1978 through 1981, Neuse and Tar

River drainages. Solid dots are positive sites, hollow dots are negative sites.

Inset shows location of Neuse and Tar River drainages in North Carolina.

Occurrence of N. lewisi in the Trent River subdrainage was first

discovered on 26 September 1978, when 35 specimens were taken fol-

lowing application of rotenone to 100 yards of the river under low-

water, no-flow conditions by the N.C. Wildlife Resources Commission.

Nine additional sites in the Trent River subdrainage, most of them with

submerged limestone outcroppings, were recorded during our survey.

20

Alvin L. Braswell and Ray E. Ashton, Jr.

Fig. 2. All known records for Necturus lewisi. Solid dots are localities sup-

ported by voucher specimens; small hollow dots are sight records believed valid;

and large hollow circles are localities supported by voucher specimens with

imprecise locality data.

Table 1 shows the capture success for N. lewisi in different category

streams. The capture success rate was significantly higher in medium

and large streams than in little and small streams. No significant differ-

ence in capture success rate between little and small streams was noted,

but a trend toward greater success in larger streams was apparent.

Frequent fluctuations in water levels caused by precipitation made

it difficult to obtain meaningful depth measurements. Therefore, no

Table 1. Comparative Necturus lewisi capture success in streams of different

relative widths (m, approx.). Numbers in parentheses are number of

sampling efforts for each stream width given; total samples = 749. Per-

centages are success rates for each stream width.

Relative stream width

Necturus lewisi Study: Distribution & Ecology 21

detailed statistical analysis for stream depth vs. capture success was

done. However, a few generalities were apparent. Capture success in

streams less than 5 1 cm deep was very poor (5.3%; 4 of 75 samples). The

shallowest stream depth recorded for a positive N. lewisi site was 31 cm.

Capture success in streams 51 to 100 cm deep was better (14.6%; 25 of

171 samples). Streams over 100 cm deep had the best capture success

rate (25%; 126 of 504 samples).

Water flow rate was recorded for 635 sampling efforts. Attempts to

obtain "normal" flow readings were hampered by fluctuating water lev-

els that changed flow patterns and rates, so no detailed statistical analy-

sis was done. The flow rate data presented in Table 2 suggest reduced

capture success in the slowest flow rate category. A lower capture suc-

cess rate in slow flowing or lentic water is supported by a lack of any

known N. lewisi collections from lakes or ponds.

Table 2. Comparative Necturus lewisi capture success at different water flow

rates. Number of sampling efforts for each flow rate category given in

parentheses. Percentages are success rates for each category.

Capture Flow rates (cm/ sec.)

success 0_io ii_20 21-30 31-40 41-50 51-60 61-70 71-80 81-90 91 +

Negative (61) (106) (96) (76) (76) (30) (14) (23)

Positive (4) (24) (35) (19) (25) (4) (4) (3)

6.2% 18.5% 26.7% 20.0% 24.8% 11.8% 22.2% 11.5%

Totals (65) (130) (131) (95) (101) (34) (18) (26) (0) (35)

Analysis of stream substrate type and capture success rate for 743

samples (Table 3) revealed a significantly better capture rate where clay

or hard soil was predominant. Capture rates were not significantly differ-

ent for the four other substrate types, although the muck and detritus

category included leaf beds, said by others to be the preferred habitat

(Brimley 1944; Fedak 1971; and Martof et al. 1980).

Data on maximum and minimum stream temperatures observed

during minnow trap sampling revealed that maximum temperatures

ranging up to 29° C had little or no bearing on trap success. However,

minimum temperatures observed during a trapping period did affect

trap success. No N. lewisi were trapped when the minimum temperature

exceeded 18° C. Significantly decreased trap success was also observed

at a minimum temperature of 0° C, when compared to success at 16° to

18° C.

The potential effects of changes in water temperature on trap suc-

cess were investigated by recording no change, general rise, or general

fall in temperature during a trapping period. Success rates of 14.8%,

20.5%, and 20.2% for no change, rise, and fall, respectively, did not

differ significantly.

22

Alvin L. Braswell and Ray E. Ashton, Jr.

Table 3. Comparative Necturus lewisi capture success on different substrates.

Number of sampling efforts for each substrate category given in paren-

theses. Percentages are success rates for each category.

Substrates

The potential effects of changes in water level on capture success

were tested by recording no change, general rise, or .general fall in water

level during a trapping period. Table 4 shows relative capture success

for each of the three catergories. Capture success rates were signifi-

cantly higher when the water levels rose than when they remained con-

stant. Capture success rates during drops in water levels did not differ

significantly from those obtained when levels were rising or when they

were constant.

Table 5 shows the relationships between capture success rates and

different levels of precipitation observed during a sampling period. No

significant differences were found.

Capture success compared to water turbidity is shown in Table 6. A

significantly higher success rate was observed in the 0-20 (Secchi disc)

category over all other categories except the 81-98 category. The differ-

ence between the 0-20 and 81-98 categories, though not significant, does

not disrupt the pattern of greater capture success in the most turbid

waters.

For examination of digestive tract contents 153 N. lewisi were

divided into two groups: 36 larvae (20-50 mm SVL) and 1 17 adults (91 +

mm SVL). Too few specimens were available in the 51 to 90 mm SVL

size range to allow comparisons. Since adults were trapped, they could

have been in a trap for 0-3 days prior to collection, and their capture of

Table 4. Comparative Necturus lewisi capture success during changing water

levels. Number of sampling efforts for each water level change in par-

entheses; percentages are success rates for each condition.

Capture Water levels

Necturus lewisi Study: Distribution & Ecology 23

Table 5. Comparative Necturus lewisi capture success for different amounts of

precipitation within a sampling period. Number of sampling efforts in

parentheses; percentages are success rates for each category.

Capture Levels of precipitation (in.)

Table 6. Comparative Necturus lewisi capture success at different levels of tur-

bidity (Secchi disc). Number of sampling efforts for each turbidity level

in parentheses; percentages are success rates for each category.

Capture Turbidity (cm)

some food items could have been trap assisted. Larval specimens were

dipnetted, thus their stomach contents may be more representative of

natural conditions. All specimens were collected from January to mid-

April and reflect feeding habits for that time interval. Initially, the two

size groups were divided into 10 mm size increments to check for

obvious size-related feeding differences. No such differences were found

within either group. The 36 larvae contained 197 food items (mean = 5.5

items/ specimen) and the 1 17 adults contained 1,435 food items (mean =

12.3 items/ specimen).

The feeding habits of adult N. lewisi (Table 7) were more diverse

than those observed in larvae (Table 8). Arthropods accounted for 99%

of the food items in larvae, but only 74.1% in adults. Ostracods and

copepods, major elements in larval diets, were not found in adults.

Adults added to their diets two groups of mollusks, several vertebrates,

various terrestrial food items (Table 10), and other aquatic organisms

too large for larvae to eat. The types of dipteran larvae eaten are listed

in Table 9. The apparent shift in types of dipterans eaten seemed to

reflect a preference for larger dipteran larvae by adults.

Data on digestive tract contents of 34 N. punctatus, collected sym-

patic with N. lewisi and divided into the same two size categories (20-

50 mm SVL larvae, N=14; 91-150 mm SVL adults, N=20), are presented

in Table 11. There was little deviation from the diet of N. lewisi. The

24 Alvin L. Braswell and Ray E. Ashton, Jr.

Table 7. Contents of digestive tracts from 1 17 adult Necturus lewisi (91-173 mm

SVL). NF = number of food items. % = percentage of total number of

food items.

Food types NF %

Mollusca

Gastropoda

Physidae

other aquatic snails

terrestrial slugs

Pelecypoda

Annelida

Oligochaeta (earthworms)

Hirudinea (leeches)

Arthropoda

Arachnida 19 1.3

Crustacea

Isopoda, aquatic

Isopoda, terrestrial

Amphipoda

Decapoda

Chilopoda (centipedes)

Diplopoda (millipeds)

Insecta

Odonata

Ephemeroptera

Plecoptera

Trichoptera

Megaloptera

Diptera*

Hemiptera

Orthoptera

Coleoptera

Dytiscidae

terrestrial beetles

terrestrial grub

Lepidoptera (caterpillars)

unidentified insects

Chordata

Fish

Anguilla

other

Amphibians (salamanders)

unidentified

Totals M35 100.0

* See Table 9 for Diptera families

Necturus lewisi Study: Distribution & Ecology 25

Table 8. Contents of stomachs from 36 larval Necturus lewisi (20-50 mm SVL).

NF = number of food items. % = percentage of total number of food

items.

Food category NF %

Annelida

Oligochaeta (earthworms) 2 1.0

Arthropoda

Crustacea

Cladocera

Ostracoda (Cypridae)

Copepoda (Cyclopidae)

Isopoda

Amphipoda

Insecta

Collembola

Odonata

Ephemeroptera

Plecoptera

Trichoptera

Diptera*

Coleoptera (Dytiscidae)

unidentified insects

Totals 197 100.0

* See Table 9 for Diptera families.

Table 9. Diptera families found in digestive tracts of larval and adult Necturus

lewisi. NF = number of food items. % = percentage of the total number

of food items for each group.

Diptera families

Simuliidae

Ceratopogonidae

Tendipedidae

Tipulidae

Chironomidae

Tabanidae

unidentified

Totals 54 27.4% 145 10.1%

26 Alvin L. Braswell and Ray E. Ashton, Jr.

Table 10. Terrestrial food items recovered from 117 Necturus lewisi digestive

tracts. These 1 10 items comprised 7.7% of the total number of items

recovered.

Slugs 7 Millipeds 2

Earthworms 48 Grasshopper 1

Spiders 19 Beetles 6

Sowbugs 8 Grub 1

Centipedes 16 Caterpillars 2

only major food difference seen in 9 larval N. lewisi collected at the

same site as the 14 larval N. punctatus was the absence of Ceratopogo-

nidae in the N. lewisi diet. Except for the absence of vertebrates, the diet

of adult N. punctatus was similar to that of adult N. lewisi.

Potential food items that seemed to be abundant in most N. lewisi

sites but were not eaten in numbers concomitant with their relative

abundance included crayfish, shrimp, amphipods, Plecoptera nymphs,

Odonata naiads, and small fish.

Subsequent to the survey an adult N. lewisi, trapped on 4 February

1984, contained an adult worm snake, Carphophis amoenus. The worm

snake passed through the salamander nearly intact; only the tail showed

signs of digestion.

DISCUSSION

Necturus lewisi is distributed widely in the Neuse and Tar River

drainages. It occupies most clean, moderate to swift flowing streams

with widths over 15.5 m in the Piedmont Plateau and along the Fall

Line. Smaller streams are less frequently inhabited and N. lewisi is not

commonly found in streams less than 5.5 m wide. Most Coastal Plain

N. lewisi sites are in or near the mainstream of the Neuse and Tar rivers

or their largest tributaries. Recent records from the mainstream of the

Neuse River, and historic records from near Washington, Beaufort

County, place N. lewisi in river areas that are close to waters occasion-

ally influenced by salt water. Their salinity tolerance, however, is

unknown.

Parts of the Trent River subdrainage of the Neuse River basin

differ from most other Coastal Plain tributaries in having carbonate

rocks. The Castle Hayne limestone belt underlies the system, and out-

croppings are common in the mainstream of the Trent River and in

many of its tributaries. Sampling efforts in the system indicate that N.

lewisi is of frequent occurrence in the area of these limestone outcropp-

ings. Outside the areas of limestone influence, Necturus punctatus

seems to replace N. lewisi in the Trent River system. How and to what

extent the limestone outcroppings relate to N. lewisi is not known. Salt

water influence and a possible lack of suitable habitat in the lower Trent

Necturus iewisi Study: Distribution & Ecology

27

Table 11. Contents of digestive tracts from 14 larval and 20 adult Necturus

punctatus collected in streams with N. Iewisi. NF = number of food

items; % = percentage of total number of food items for each size group.

Necturus punctatus

Food items

Mollusca

Gastropoda

Pelecypoda

Annelida

Oligochaeta

Arthropoda

Arachnida (pseudoscorpion)

Crustacea

Cladocera

Ostracoda

Copepoda

Isopoda

Amphipoda

Chilopoda

Insecta

Ephemeroptera

Plecoptera

Trichoptera

Megaloptera

Diptera

Simuliidae

Ceratopogonidae

Tendipedidae

Anthomyiidae

Tipulidae

Chironomidae

unidentified

Hymenoptera

Coleoptera (Dytiscidae)

Lepidoptera (larva)

unidentified

Unidentified arthropod

Totals

1.7

12.3

1.0

57

100%

Mean # food items/ specimen

4.1

5.0

28 Alvin L. Braswell and Ray E. Ashton, Jr.

and Neuse rivers may be acting to isolate the N. lewisi in the Trent

River subdrainage from the main population in the Neuse River basin.

Regrettably, water fluctuations made it impossible to record stream

widths, depths, and flow rates under stable conditions for most collect-

ing sites. A stream index based on such measurements would likely be

most valuable if the measurements were taken under dry-weather,

summer conditions when oxygen levels are more critical for N. lewisi.

Under prevailing winter and spring conditions, stream width was the

most reliable of the three measurements and was used to formulate the

stream categories in Table 1. Stream size appears to be a limiting factor

for N. lewisi occurrence only in the case of small streams. Cursory

observations on Piedmont streams indicate that N. lewisi does not occur

in streams where water flow ceases under normal summer dry-weather

conditions. Based on our data, N. lewisi seems to survive better in

streams wider than 15 m, deeper than 100 cm, and with a main channel

flow rate greater than 10 cm/ sec. This assessment agrees with Hecht's

(1958) general theory of the streams N. lewisi occupies.

Although capture success indicates that N. lewisi prefers clay or

hard soil substrate, a variety of conditions appear suitable (Table 3).

Siltation levels may be a more critical factor, since heavy siltation could

reduce the availability of food and cover, and hinder reproduction by

smothering nests and eggs. Generally, siltation seemed light to moderate

in most N. lewisi sites.

Capture success rates do not support the contention of Fedak

(1971) and Martof et al. (1980) that N. lewisi spends the winter in leaf

beds. That hypothesis probably arose because N. lewisi can be captured

regularly by seine and dipnet only when they are in such habitat (see

Ashton, this publication). However, too much speculation about habitat

preferences based on trapping success is unwarranted, since the distance

N. lewisi will travel to a baited trap is unknown.

A few places where N. lewisi was expected but not collected during

our study include: the Neuse River from east of Raleigh to the vicinity

of Smithfield; Swift Creek in Johnston County; and the Tar River from

Rocky Mount to about 20 km downstream. To what extent municipal

and industrial effluents may be acting to cause such apparent gaps in

the distribution is unknown.

The Little River from eastern Wake County to the vicinity of

Goldsboro, and the Trent River in Jones County, appear to have the

best N. lewisi populations in the Neuse River drainage. Virtually the

entire Tar River from Granville County to central Pitt County, with the

exception of Rocky Mount Reservoir and the 20 km section below

Rocky Mount, appears to have a healthy population. Other Tar River

drainage streams with apparent good populations include Fishing and

Necturus lewisi Study: Distribution & Ecology 29

Little Fishing creeks in Warren and Halifax counties, and Swift Creek

in Nash County.

Comparing trap capture success to factors such as water levels,

stream temperatures, precipitation, turbidity, time of day, and season

provides some information on activity patterns. Activity away from

cover and consequent capture in traps usually occurs at night and when

stream temperature ranges between 0° C. and 18° C. Temperature

changes within this range seem to have little effect on activity.

Although precipitation seems to have no direct effect on activity,

the rising water levels and high turbidity it produces show a positive

correlation with increased activity. The "cover" that turbidity provides,

and increase in terrestrial food items produced by runoff and rising

water, may trigger greater activity under these conditions.

The difficulty encountered in trapping N. lewisi during warm and

hot seasons is currently inexplicable, but other authors have suggested it

may relate to activity patterns of predatory fish. Neill (1963) and Shoop

and Gunning (1967) alluded to this possibility to explain the activity

patterns observed in N. beyeri and N. maculosus louisianensis, which

are similar to those seen in N. lewisi. Fish migrations and spawning

activity markedly increase in April when minimum stream temperatures

routinely exceed 18 degrees C. Thus, this temperature may represent a

threshold above which N. lewisi cannot forage abroad without serious

threat from predators. There are, however, alternative hypotheses. For

one thing, warming water correlates with egg deposition and subsequent

nest guarding by N. lewisi (Ashton and Braswell 1979). In addition,

April and May are months in which potential prey such as crayfish,

other invertebrates, and fish increase in number due to recruitment of

young into their populations. A greater supply of food could greatly

reduce foraging activities. Shoop and Gunning (1967) indicated that

crayfish of the genus Procambarus were the primary food of N. macu-

losus and N. beyeri in Louisiana. (See Cooper and Ashton, this publica-

tion, for information on crayfish associates of N. lewisi in the study

areas.)

Analysis of digestive tract contents of adult N. lewisi shows that a

wide variety of aquatic invertebrates, aquatic vertebrates, and terrestrial

invertebrates are acceptable food items. The diet of larvae is largely

restricted to aquatic invertebrates, mostly arthropods. The size of the

prey item that a certain size class of salamander can accomodate seems

to be the most important factor in prey selection.

Prey consumed by adults indicates that they visit a broad range of

habitats on feeding forays. Prey items that are routinely found in flow-

ing and slack waters, in leaf beds, under logs and rocks, and in sand and

gravel, were present in their digestive tracts.

30 Alvin L. Braswell and Ray E. Ashton, Jr.

Virtually all larval specimens were collected by netting in leaf beds,

and their representative diet may be biased in favor of prey available in

the beds. Whether larvae are more dependent on leaf beds than are

adults, or whether collecting techniques currently used are inadequate to

collect them elsewhere, is currently unknown. As previously stated,

adults were once thought to rely heavily on leaf beds.

Necturus lewisi, then, is an opportunistic feeder whose diet proba-

bly varies from season to season as changes in composition and abun-

dance of the prey community occur. The data presented here should

indicate their diet from January to mid-April, but additional investiga-

tion is needed to determine just how much seasonal variance their diet

displays.

The abundance of certain potential prey in N. lewisi sites, and the

low incidence of these items in digestive tract contents, probably reflect

the difficulty encountered by the salamanders in capturing certain spe-

cies. Examples of abundant and highly mobile potential prey are cray-

fish, palaemonid shrimp, amphipods, plecoptera nymphs, odonate nai-

ads, and small fish.

The great similarity in diets of N. lewisi and N. punctatus would

certainly seem to encourage competition between these two species,

especially in sites where food is scarce. However, the year-round diet of

N. punctatus is no better known than that of N. lewisi. Also, N. puncta-

tus routinely exploits smaller Coastal Plain streams than N. lewisi does;

the major area of sympatry between them is along the Fall Line Zone.

Investigation of the habitats and habits of N. punctatus in adjacent

drainages where N. lewisi is absent could reveal more about competition

between the two species. Necturus punctatus may possess greater toler-

ances for oxygen and temperature ranges, ph levels, and drought

conditions.

The wide distribution of N. lewisi in the Neuse and Tar River

drainages argues against Endangered or Threatened status at this time.

However, a status of Special Concern may be warranted due to N. lewi-

si's need for large streams with relatively clean, flowing water. Stephan

(1977) suggested that N. lewisi be considered a species of Special Con-

cern, based on its restricted distribution and susceptibility to environ-

mental degradation. Some distributional gaps that may be attributable

to effluents were previously indicated. Large reservoirs and stream

channelization cause additional loss of suitable habitat. The increasing

industrial and municipal growth in areas influencing the Neuse and Tar

River drainages may have a serious impact on N. lewisi, as well as other

unique species with similar habitat requirements, unless informed mea-

sures are taken to adequately protect their riverine environment.

Necturus lewisi Study: Distribution & Ecology 31

ACKNOWLEDGMENTS.— The efforts of several individuals war-

rant credit and special thanks. Field technicians Jerry Reynolds, Angelo

Capparella, Eric Rawls, Roger Mays, and Paul Freed weathered all sorts

of misfortune to collect Necturus. John E. Cooper and William M.

Palmer provided comments on the manuscript and assisted in many

other ways. John Clamp and Jerry Reynolds identified most of the

invertebrates from digestive tracts. North Carolina Wildlife Commission

personnel provided specimens and information resulting from their

stream survey work. Additional specimens were received from Gary

Woodyard, Sidney Shearin, and Perry Rogers. Jack Nealon (USDA)

and Mary Jo Gilliam assisted with the statistical analysis. Computer ser-

vices and personnel of the N.C. Department of Agriculture were very

helpful. Several institutions, previously listed, graciously provided

information on their N. lewisi holdings. This project was partially sup-

ported by Title 6 funds granted to the state of North Carolina through

the Cooperative Agreement between the U.S. Fish and Wildlife Service

and the N.C. Wildlife Resources Commission. Additional funding was

provided by the N.C. State Museum of Natural History, N.C. Depart-

ment of Agriculture.

LITERATURE CITED

Ashton, Ray E., Jr., and A.L. Braswell. 1979. Nest and larvae of the Neuse

River Waterdog, Necturus lewisi (Brimley) (Amphibia: Proteidae). Brim-

leyana 7:15-22.

Behler, John L., and F.W. King. 1979. The Audubon Society Field Guide to the

North American Reptiles and Amphibians. Alfred A. Knopf, New York.

719 pp.

Bishop, Sherman C. 1943. Handbook of Salamanders. Comstock Publishing Co.,

Ithaca, xiv + 555 pp.

Brimley, C.S. 1896. Batrachia found at Raleigh, N.C. Am. Nat. 30:500-501.

1915. List of reptiles and amphibians of North Carolina. J. Elisha

Mitchell Sci. Soc. 30(4):3-14.

1920. Notes on Amphiuma and Necturus. Copeia 1920(77):5-7.

1924. The water dogs {Necturus) of North Carolina. J. Elisha Mit-

chell Sci. Soc. 40(3 & 4): 166-168.

_. 1944. Amphibians and reptiles of North Carolina. Carolina Biologi-

cal Supply Company, Elon College, N.C. Reprinted from Carolina Tips

(1939-43). 63 pp.

Conant, Roger. 1975. A Field Guide To Reptiles and Amphibians of Eastern

and Central North America. Houghton Mifflin Co., Boston, xviii + 429 pp.

Fedak, Michael A. 1971. A comparative study of the life histories of Necturus

lewisi Brimley and Necturus punctatus Gibbes (Caudata: Proteidae) in

North Carolina. Unpubl. Master's Thesis, Duke Univ., Durham, N.C. 103 pp.

Hecht, Max K. 1953. A review of the salamander genus Necturus Rafinesque.

Ph.D. Dissert., Cornell Univ., Ithaca. 248 pp.

32

Alvin L. Braswell and Ray E. Ashton, Jr.

1958. A synopsis of the mud puppies of eastern North America.

Proc. Staten Island Inst. Arts Sci. 2/(l):3-38.

Martof, B.S., W.M. Palmer, J.R. Bailey and J.R. Harrison, III. 1980. Amphi-

bians and Reptiles of the Carolinas and Virginia. Univ. North Carolina

Press, Chapel Hill. 264 pp.

Neill, Wilfred T. 1963. Notes on the Alabama waterdog, Necturus alabamensis

Viosca. Herpetologica 79(3): 166-174.

Shoop, C. Robert, and G.E. Gunning. 1967. Seasonal activity and movements

of Necturus in Louisiana. Copeia 1967(4):732-737.

Stephen, D.L. 1977. Necturus lewisi Brimley. Neuse River Waterdog. Pp. 317-

318 in J.E. Cooper, S.S. Robinson and J.B. Funderburg (eds.). Endangered

and Threatened Plants and Animals of North Carolina. N.C. State Mus.

Nat. Hist., Raleigh, xvi + 444 pp.

Viosca, Percy, Jr. 1937. A tentative revision of the genus Necturus with descrip-

tions of three new species from the southern gulf drainage area. Copeia

1937(2): 120- 138.

Yarrow, H.C. 1882. Check list of North American Reptilia and Batrachia. Bull.

U.S. Natl. Mus. 24:1-249.

APPENDIX A

Necturus lewisi Localities

Neuse River Drainage

County

Stream

Locality

4.8 km ENE Tuscarora

7.6 km NNE Cove City

4.0 km NE Fort Barnwell

7.2 km ESE Fort Barnwell

7.2 km SW Askin

7.6 km E Jasper

Durham

12.9 km NE Durham

5.6 km SE Bahama

14.9 km NNE Durham

10.5 km NNE Durham

6.8 km SW Walstonburg

8.4 km ESE Hookerton

3.2 km SE Hookerton

5.0 km N Princeton

6.8 km SSW Kenly

4.8 km WSW Kenly

6.0 km W Kenly

16.1 km NW Kenly

18.5 km ENE Clayton

18.5 km NE Clayton

18.8 km NE Clayton

7.9 km WNW Kenly

Source/ Voucher

NCSM 22279

DU A9129

NCSM 22282-84

NCSM 22289-94

NCSM 17557

DU A 13446-50

FMNH 84008

AMNH 32078

NCSM 7

NS**

NCSM 22475

NCSM 22227

NS**

NCSM 21935-37

NCSM 21663-73

NS**

NCSM 21939

NCSM 21940

NCSM 21674,21941-49

NCSM 21675,21957-61

NCSM 22119-34

NCSM 22156

NCSM 21676-77

NCSM 22181

NCSM 22182-85

Necturus lewisi Study: Distribution & Ecology

33

Wake

Neuse R.

5.0 km W Smithfield

13.7 km SE Four Oaks

19.3 km ESE Benson

10.9 km SSW Princeton

10.1 km NW Trenton

8.9 km WNW Trenton

0.8 km ESE Pollocksville

1.6 km N Trenton

6.4 km WNW Trenton

7.6 km NW Trenton

4.8 km NW Pollocksville

1.2 km NW Comfort

7.2 km NNE Comfort

4.0 km SSE Pleasant Hill

Kinston

2.0 km SE Grifton

1.2 km NW Grifton

8.4 km SSW LaGrange

9.3 km SSE LaGrange

8.4 km NE Kinston

5.6 km SE Kinston

10.1 km SE Hillsborough

4.0 km ENE Hillsborough

8.0 km E Hillsborough

8 km NW Hillsborough

5.3 km SSE Timberlake

4.8 km S Bell Arthur

Raleigh

near Raleigh

Raleigh

10.5 km NW Raleigh

Wendell

4.4 km NNE Wendell

6.4 km E Rolesville

3.6 km SW Zebulon

6.0 km E Rolesville

5.6 km E Rolesville

4.4 km WNW Zebulon

5.1 km ENE Zebulon

Raleigh

NCSM 22245

NCSM 21678

NCSM 21679

NCSM 22222

NS**

NCSM 22266

NS**

NCSM 22276

NCSM 22267-68

NCSM 22261-64

NCSM 22265

NCSM 22269-75

NCSM 19831

NCSM 22763

NCSM 22257-58

USNM 8348

NCSM 22298

NCSM 22299

NCSM 21904-08

NCSM 21909-10

NCSM 21911-14

NCSM 21915

NCSM 22229-31

NCSM 9920-21

DU A8442-46*

DU A2003

NCSM 22226

NCSM 22295

NCSM 2, 12, MCZ5877

NCSM 4

MCZ 17729

NCSM 22480,22515

AMNH 41788, 45306

FMNH 84009

NCSM 22762

DU A6259*

NCSM 16551-56*

NCSM 4789

DU A6700*

NCSM 19826*

DU A8920

DU A8997

EEB 6641-42

NCSM 5, 20

FMNH 84010

UF/FSM 1120

CM 10594

MCZ 17726-27

USNM 73848

TU 6157

NCSM 10-11

NCSM 13-19

NCSM 15052

NCSM 16550

NCSM 22228, 22232-35

NCSM 22481-500*

NCSM 22508-12

NCSM 19363

34

Alvin L. Braswell and Ray E. Ashton, Jr.

Tar River Drainage

County Stream

Locality

Source/ Voucher

Beaufort

Edgecombe

Franklin

Granville

Halifax

Nash

Tranters Cr.

Fishing Cr.

Hendricks Cr.

Swift Cr.

Tar R.

Town Cr.

Cedar Cr.

Crooked Cr.

Cypress Cr.

Sandy Cr.

Shocco Cr.

Tar R.

Fishing Cr.

Little Fishing Cr.

Peachtree Cr.

Red Bud Cr.

Stoney Cr.

Washington

Tarboro bridge

5.3 km ENE Leggett

5.3 km NNE Whitakers

1.0 km S Tarboro

10.0 km NNW Tarboro

7.9 km E Pinetops

1.2 km SSE Tarboro

2.9 km NNW Tarboro

4.3 km ENE Pinetops

6.8 km S Louisburg

6.0 km SW Louisburg

2.9 km S Bunn

3.2kmNW Bunn

6.9 km ESE Bunn

6.3 km SE Centerville

3.1 km SW Centerville

14.5 km NNE Louisburg

2.7 km N Centerville

8.0 km NW Louisburg

4.8 km NNE Bunn

4.8 km ESE Bunn

2.9 km ENE Bunn

5.6 km N Wilton

6.0 km N Wilton

10.9 km SSW Oxford

10.3 km SW Oxford

10.1 km WSW Oxford

13.0 km W Oxford

1.4kmSW Berea

5.5 km NW Berea

7.4 km WSW Enfield

12.9 km WSW Enfield

7.6 km SSE Hollister

8.7 km SW Hollister

8.4 km SE Hollister

12.4 km SSE Hollister

4.4 km ESE Hollister

8.9 km N Hollister

4.5 km E Hollister

11.7 km WNW Nashville

4.2 km NNE Castalia

7.4 km WNW Nashville

1.8 km W Nashville

NCSM 1

NCSM 6

USNM 7015

NCSM 22386-87

NCSM 22393,22638

NCSM 22380

NCSM 22384

NCSM 22367-68

NCSM 22369

NCSM 22370

NCSM 22365-66

DU A9510, NCSM 22304

DU A8896, A8999

NCSM 22312

NCSM 22313-14

NCSM 22317

NCSM 22308-11

NCSM 22315

NCSM 22331

NCSM 22316

NCSM 22300-01

NCSM 22302-03

NCSM 22305

NCSM 22306

NCSM 22321

NCSM 22322

NCSM 22323

NCSM 22324, 22637

NCSM 22325-26

NCSM 22327

NCSM 22328-29*

NCSM 22330

NCSM 22395

NCSM 22396

NCSM 22403

NCSM 22404

NLSM 22399-400

NCSM 22401

NCSM 19866, 19904-07

NCSM 22402

NCSM 22458-60

NCSM 22344

NCSM 22354-61

NCSM 22408

NCSM 22409

Necturus lewisi Study: Distribution & Ecology

35

6.4 km NE Castalia

5.1 kmNE Red Oak

10.5 km NNW Red Oak

3.6kmSW Rocky Mt.

12.9 km SSW Nashville

3.7 km SW Spring Hope

14.1 km S Nashville

at (near) Falkland

3.2 km NNW Falkland

2.8 km NNE Falkland

1.6 km E Bruce

4.8 km E Greenville

5.6 km NW Greenville

1.6 km ESE Falkland

1.6 km SE Falkland

5.8 km S Kittrell

7.2 km SW Kittrell

6.8 km ESE Inez

16.6 km SE Warrenton

4.5 km N Inez

7.1 km SSE Warrenton

4.7 km SSE Warrenton

2.9 km SW Warrenton

15.7 km ESE Warrenton

8.4 km SE Vaughn

6.3 km NE Areola

9.5 km ESE Warrenton

14.2 km SSE Warrenton

NCSM 22405

NCSM 22406

NCSM 9922-24

DU A8887-95*

NCSM 22371

NCSM 22372-74

NCSM 22375-78

DU A9603

NCSM 22379

TU 19081(2)

NCSM 22411

NCSM 22444

NCSM 22445-46

NCSM 22447-51

NCSM 22452

NCSM 22426-28

NCSM 22438-39

NCSM 22319

NCSM 22320

NCSM 22332

NCSM 22333

NCSM 22334

NCSM 22340

NCSM 22341

NCSM 22342

NCSM 22336-37

NCSM 22338

NCSM 22335

NCSM 22339

NCSM 22318

♦Additional catalogued but unlisted specimens are available from this locality.

**Specimens collected during Necturus survey not catalogued for this site.

Accepted 10 August 1984

Chromosome Evolution in Salamanders of

the Genus Necturus

Stanley K. Sessions

Museum of Vertebrate Zoology,

University of California, Berkeley, California 94720

AND

John E. Wiley

St. Paul's College,

Box 751, Lawrenceville, Virginia 23868

ABSTRACT. — All species of the salamander genus Necturus (N. ala-

bamensis, N. beyeri, N. lewisi, N. maculosus, and N. punctatus) have

19 pairs of chromosomes (2n = 38), and well-differentiated hetero-

morphic sex chromosomes of the XY male/ XX female type. Four dis-

tinct karyotypes are observed among the species in terms of C-band pat-

terns, the proportion of asymmetrical chromosomes such as telocen-

trics, and the degree of differentiation of the sex chromosomes. Nectur-

us beyeri appears to be identical to N. maculosus in these features

while the three other species have uniquely different karyotypes; N.

alabamensis may be polymorphic for an intermediate number of telo-

centric chromosomes. Interspecific homologies between the most

asymmetrical chromosomes, including telocentrics, are suggested by

the similar position of these chromosomes in the karyotypes and sim-

ilarity in C-band patterns. The species appear to exhibit sequential

stages of karyological differentiation in the order: lewisi-punctatus-

alabamensis-beyeri + maculosus. Karyology correlates with geographic

distribution in a simple pattern suggesting that gradual karyological

differentiation occurred as populations became established southward

along the Coastal Plain of southeastern United States, around the

southern end of the Appalachian Mountain Range, and into the Mis-

sissippi River drainage system. Thus, N. lewisi represents the most

primitive form and N. maculosus the most derived condition.

INTRODUCTION

Salamanders of the genus Necturus of eastern North America have

been used extensively for biological research for well over 100 years, but

the phylogenetic relationship of Necturus to other salamanders, and sys-

tematics within the genus, remain problematical. Necturus represents an

ancient lineage of neotenic (sensu Gould 1977), permanently aquatic

salamanders, with a generalized larval morphology that obscures affini-

ties to other living salamanders (Hecht 1957). Most workers consider

Necturus to be most closely related to the European blind salamander,

Proteus anguinus, and place both genera in the same family, Proteidae

(Brandon 1969; Larsen and Guthrie 1974; Naylor 1978; Noble 1931;

Brimleyana No. 10:37-52. February 1985. 37

38 Stanley K. Sessions and John E. Wiley

Morescalchi 1975). Kezer et al. (1965) presented karyological evidence

that supports this view. Hecht and Edwards (1976) reviewed the system-

atic data for these groups of salamanders and concluded that morpho-

logical information is not sufficient to resolve this problem, and that

more biochemical and karyological studies are needed.

One of the most thorough treatments to date of the genus Necturus

is that of Hecht (1958), who recognized four species on the bases of

external morphology, ontogeny, and geographic distribution: Necturus

beyeri Viosca, Necturus lewisi (Brimley), Necturus maculosus (Rafi-

nesque), and Necturus punctatus (Gibbes). He also tentatively recog-

nized a subspecies of N beyeri, N b. alabamensis Viosca, but described

it as "one of the most distinct forms in the genus" (Hecht 1958:17), and

seemed somewhat ambivalent as to its taxonomic status. Subsequently

Brode (1970) revised the genus, mainly on osteological grounds, recog-

nizing two species, TV. maculosus and N punctatus, and six subspecies:

TV. m. maculosus, N m. lewisi, N m. walkeri, N p. punctatus, N p.

alabamensis, and TV. p. beyeri. The present systematic status of this

group of salamanders is one of uncertainty and disagreement, mostly

because there are so few distinguishing morphological features between

the various forms. Nevertheless, most recent workers recognize five

species — TV. alabamensis, N beyeri, N lewisi, N maculosus, and TV.

punctatus — as well as several subspecies of TV. maculosus (Brame 1967;

Gorham 1974; Conant 1975).

All species of Necturus, with the exception of TV. maculosus, are

distributed along the Coastal Plain of southeastern United States, from

southeastern Virginia to eastern Texas (Fig. 1). Necturus maculosus is

by far the most widely distributed species, with a range extending fan-

like from an apex in Louisiana and broadening northward to southeast-

ern Manitoba in the west and southeastern Quebec in the east, essen-

tially encompassing the entire Mississippi River drainage system. The

combined ranges of these species result in a more-or-less continuous

distribution of Necturus over most of eastern North America, inter-

rupted in the east by the Appalachian Mountains which form a wedge

separating the two coastal species, TV. lewisi and TV. punctatus, from

inland populations of TV. maculosus. According to Brode (1970), partial

sympatry and morphological intergraduation may occur between TV.

punctatus and TV. alabamensis, and between the latter form and TV.

beyeri. Necturus lewisi and TV. punctatus, however, are the only species

definitely known to occur in sympatry, and are morphologically the

most distinct of all Necturus species (Hecht 1958).

Results of a recent electrophoretic analysis by Ashton et al. (1980)

suggest that at least three species, TV. maculosus, TV. lewisi, and TV. punc-

tatus, are distinct, long-isolated species. Only TV. maculosus has been

studied karyologically (Seto et al. 1964; Morescalchi 1975; Sessions

Necturus Chromosome Evolution 39

1980); neither N. beyeri nor N. alabamensis have been so examined.

Relative to other salamanders, N. maculosus has a karyotype that is

distinctive in chromosome number and morphology, and particularly in

the degree of differentiation of the sex chromosomes (Sessions 1980).

Our study was carried out to ascertain the degree to which karyological

changes have accompanied diversification and divergence within the

genus Necturus, and to elucidate the relationship between the geogra-

phic distribution and the evolutionary history of this group of sala-

manders.

MATERIALS AND METHODS

The following specimens were used in this study and unless other-

wise indicated all have been deposited in the Museum of Vertebrate

Zoology, University of California, Berkeley. Abbreviations used below:

ASU = Department of Biology, Appalachian State University, Boone,

NC; CR = county road; SR = state road.

Necturus alabamensis.— 2$$, 19, Black Creek at SR 20, 1.6 km

W of Bruce, Walton Co., FL; 20 May 1980; 1$, Juniper Creek,

approx. 1.6 km S of Juniper, 137 m elevation, Marion Co., GA; 30

November 1980.

Necturus beyeri. — 19, 1$ (deposited at ASU), approx. 24 km S

of Nacogdoches on Bernaldo Creek, Stephen F. Austin State Univer-

sity Experimental Forest, Nacogdoches Co., TX; 29 March 1981.

Necturus lewisi. — \$, 19, Tar River along State Hwy 44, 2.9 km

NNW of Tarboro, Edgecombe Co., NC; January 1980; 1#, Tar River,

14.1 km S of Nashville, Nash Co., NC; January 1980.

Necturus maculosus. — 19, Wisconsin (exact locality not availa-

ble); from Carolina Biological Supply Co.

Necturus punctatus. — 3##, Little River, crossing of CR 2224,

Wake Co., NC; August and January, 1981.

Mitotic chromosomes were prepared from colchicine-treated intes-

tinal epithelium and spleen, following the technique described in Kezer

and Sessions (1979). Air and flame dried slides were also made from

peripheral blood after in vivo treatment with phytohemagglutinin (PHA,

Sigma) and Colcemid (Sigma). C-banding was carried out on squash

preparations of intestinal epithelium and spleen cells, using the tech-

nique of Schmid et al. (1979). At least three mitotic cells were examined

from each individual. Idiograms were constructed from measurements

of a single representative mitotic spread for N. alabamensis, N. lewisi,

N. maculosus, and N. punctatus.

RESULTS

All species of Necturus studied have 19 pairs of chromosomes (2n =

38), including a pair of well-differentiated heteromorphic sex chromo-

40 Stanley K. Sessions and John E. Wiley

somes of the XY male/ XX female type. In terms of chromosome mor-

phology and degree of differentiation of the X and Y sex chromosomes,

we discern at least four unique karyotypes in the genus (Fig. 2a-d).

These are represented by idiograms in Figure 3.

Necturus lewisi has a completely bi-armed karyotype (Figs. 2a, 3;

Table 1), and the X-chromosome is a medium-size metacentric so that

the sex chromosome pair is in position 6 of the karyotype. The Y-

chromosome of this species is subtelocentric and mostly euchromatic

(unstained) after C-banding, except for two bands of heterochromatin,

one light and one dark, in the middle of the long arm, and the proximal

portion of the long arm which is lightly stained (Fig. 4). Two chromo-

somes (besides the Y-chromosome) are extremely asymmetrical (subte-

locentric) with large arm ratios (Fig. 3; Table 1).

Necturus punctatus also lacks telocentric chromosomes (Figs. 2b, 3;

Table 1), but its X-chromosome is larger than that of N. lewisis and the

sex chromosomes are in position 3 or 4 of the karyotype. Necturus

punctatus has three pairs of subtelocentric chromosomes (besides the

Y-chromosome), two medium-size and one small, with large arm ratios

(Table 1). The medium-size subtelocentrics of N. punctatus are in sim-

ilar positions in the karyotype to the most asymmetrical chromosomes

of N. lewisi (Fig. 3). The subtelocentric Y-chromosome of N. punctatus

is considerably more heterochromatic than the Y-chromosome of N.

lewisi, but less heterochromatic than the Y-chromosome of N. maculo -

sus (Fig. 4).

We have found at least two and possibly three different kary-

omorphs in specimens collected from the range of N. alabamensis,

which differ in the number of telocentric chromosomes. The karyotype

of a female from Georgia is identical to that of N. maculosus, with 12 (6

pairs) telocentric chromosomes. A male and female from Florida, on

the other hand, have karyotypes with only 8 (4 pairs) telocentric chromo-

somes (Fig. 2c). A third specimen from the same locality in Florida

may have 10 (5 pairs) telocentrics, although this count was based on

three mitotic preparations of mediocre quality. A male specimen with

eight telocentrics was used for the construction of an idiogram (Fig. 3).

In this karyotype, two of the telocentric chromosomes are medium-size

and two are small (Table 1; Fig. 3). The medium-size telocentrics are in

positions in the karyotype similar to the most asymmetrical medium-

size chromosomes in N. lewisi and Ar. punctatus, suggesting interspecific

homology of these chromosomes (Fig. 3). The sex chromosomes of this

specimen fall into position 3 of the karyotype (Fig. 3), as in N. maculo-

sus (Sessions 1980; this paper). In addition, the subtelocentric Y-

chromosome is more heterochromatic with a more complex banding

pattern than in N. punctatus, but is somewhat less heterochromatic than

the Y-chromosome of N. maculosus (Fig. 4).

Necturus Chromosome Evolution

41

N. alabamensis

N. punctatus

Fig. 1. Map showing approximate distribution of species of the genus Nectu-

rus used in this study (adapted from Conant 1975).

Specimens of N. beyeri from Texas have identical karyotypes to

that of N. maculosus, with 12 (6 pairs) telocentric chromosomes (Fig.

5). As in the other species, two of these pairs of asymmetrical chromo-

somes are medium-size, and the remaining telocentrics are small. The

Y-chromosome of N. beyeri is also virtually identical to that of N.

maculosus.

A karyotype and idiogram of N. maculosus were presented earlier

(Sessions 1980), but a new C-band idiogram of this species was con-

structed for this study (Fig. 3). It shows the position of the sex chromo-

somes and the 6 pairs of telocentric chromosomes in the karyotype. The

subtelocentric Y-chromosome of this species is more heterochromatic

than that in any of the other species of Necturus (Fig. 4).

DISCUSSION

Our karyological data support previously reported electrophoretic

evidence (Ashton et al. 1980) that N. lewisi, N. punctatus, and N. macu-

losus are distinct, probably long-isolated species. These data further

suggest that N. alabamensis, which has not been studied electrophoreti-

cally, may represent another distinct species, and that N. beyeri is only a

little-differentiated southern form of N. maculosus. Karyological evi-

42

Stanley K. Sessions and John E. Wiley

--..,.•*. .•""

'''J?/

:'■ ;- ■ , f i,'*

^^tm

Jr. J» < '*.??*d * L \ w v^4..

• I #0*

i 1

M

i ^ «

it

Fig. 2a-d. C-banded mitotic preparations of Necturus species: a, N. lewisi; b,

W. punctatus; c, JV. alabamensis (8 telocentrics); d, iV. maculosus. Arrows indi-

cate Y-chromosomes. Scale = 10 micrometers.

dence is also in accord with electrophoretic data in that N. lewisi and N.

punctatus are more similar to each other than either is to N. maculosus.

The karyological information, however, reveals additional details of

interrelationships that have not been resolvable by the electrophoretic

analysis.

While all species of Necturus have the same diploid number of

chromosomes, they differ in the degree of karyotype asymmetry (Table

2). The species form two groups on the basis of presence or absence of

telocentric chromosomes: N. lewisi and N. punctatus have completely

bi-armed karyotypes, while N. maculosus, N. beyeri, and N. alabamen-

sis all have several pairs of telocentric chromosomes. In terms of

Necturus Chromosome Evolution

43

10

QfiSs e

§s

SSSSBSsss

12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

B

10 \

fiflSUSQ

55§s

10

X

X i

S^gft

SB8§sass

0

D

10

%

XX

12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Fig. 3. Idiograms of Necturus species showing four kinds of karyotypes found

in the genus. A, N. lewisi; B, N. punctatus; C. N. alabamensis; D, N. maculosus.

Dark regions and lines represent C-band heterochromatin. Stippled areas on

Y-chromosomes represent light staining C-band heterochromatin. Presumed

homologous chromosomes are connected by dashed lines between idiograms.

44

Stanley K. Sessions and John E. Wiley

L P A M

Fig. 4. Diagrammatic representation of the four kinds of Y-chromosomes

found in Necturus. L-N. lewisi, P = N. punctatus, A = N. alabamensis, M = N.

maculosus. Light, stippled, and dark regions represent euchromatin, lightly

stained C-band heterochromatin, and darkly stained C-band heterochromatin,

respectively.

Fig. 5. Mitotic, unstained chromosome spread of a female N. beyeri showing

12 telocentric chromosomes. Two bi-armed chromosomes were squashed away

from the main spread and are not included. Scale = 10 micrometers.

Necturus Chromosome Evolution

45

71

.1

v \J

->

-\m

u

A

4d2j

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V

T

Fig. 6. Map showing relationship between geographic distribution and phy-

logenetic history in the genus Necturus. Karyological differentiation occurred as

populations of Necturus become established increasingly southward along the

coastal plain east and south of the Appalachian Mountains (designated by

hatching). Eventual arrival at the Mississippi River drainage system allowed

explosive northward dispersal of the karyologically most derived form, N.

maculosus.

chromosome asymmetry, sex chromosome differentiation, and /or pro-

portion of telocentric chromosomes, N. punctatus and N. alabamensis

have seemingly intermediate karyotypes. Since these two species also

have a somewhat intermediate geographic distribution, we hypothesize

that karyological evolution within the genus involved progressive differ-

entiation of the X and Y sex chromosomes, through increasing hete-

rochromatinization of the Y-chromosome and increasing relative size of

the X-chromosome, with a concomitant increase in the degree of

chromosomal asymmetry through pericentric inversion or some other

46

Stanley K. Sessions and John E. Wiley

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cu »—i

o is

< 3 c« ™

£

o

S <*

£

o

Necturus Chromosome Evolution

47

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- 8

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O <n rt

r*"i — O

(J\ \D CI

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en — « ©

ITi >0 Tt

W-) CO Tt

ro — ' O

in

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48 Stanley K. Sessions and John E. Wiley

Table 2. Summary of chromosome morphology of four species of Necturus,

from Table 1 (disregarding subtelocentric Y-chromosomes).

Chromosome morphology

kind of non-Robertsonian mechanism of centromere shifts. This latter

process may represent an example of the phenomenon referred to by

White (1973) as "karyotypic orthoselection," though it is not clear how

selection could be involved in this case.

We consider the karyotype of N. lewisi to represent the primitive

condition within the genus, since this species has the least differentiated

sex chromosomes. Necturus lewisi has the most restricted range of any

Necturus and may represent a relict.

Necturus punctatus has more differentiated sex chromosomes than

N. lewisi; the X-chromosome is larger relative to the Y-chromosome,

and the Y-chromosome is more heterochromatic. In this species, all

chromosomes are bi-armed, but they show more asymmetry in centro-

mere position than is seen in N. lewisi (Table 2). The similar sizes and

C-band patterns of the most asymmetrical chromosomes in both species

suggest that they are homologous chromosomes (Fig. 3). The geogra-

phic distribution of N punctatus includes that of N. lewisi, but is much

larger, extending southward to possibly overlap with the range of N alabam-

ensis in the southern Gulf states (Fig. 1).

Necturus alabamensis has more highly differentiated sex chromo-

somes than N punctatus, in terms of Y-chromosome heterochromatin,

and an even more asymmetrical karyotype with at least four pairs of

telocentric chromosomes (Table 2; Fig. 3). This species is found south of

the southern limit of the Appalachian mountain range and, with TV.

beyeri, is located at the southern end of the range of N maculosus (Fig.

1). Necturus beyeri appears to be identical to N maculosus in number

of telocentrics and in Y-chromosome differentiation.

At least two and possibly three karyotypes were encountered in

specimens collected in the geographic range of N alabamensis, with 8,

10, and 12 telocentrics. Several interpretations of this apparent kary-

ological variability are possible. Perhaps N alabamensis represents a

karyological intergrade between TV. punctatus and TV. beyeri. If this is

so, then heterozygotes with heteromorphic telocentric/ bi-armed chrom-

osome pairs should occur. Such heterozygotes, however, were not found

Necturus Chromosome Evolution 49

in this investigation. Furthermore, the Y-chromosome of the 8-telocentric

male N. alabamensis differs in its C-band pattern from both N. puncta-

tus and N beyeri, suggesting that it is a distinct form. Although C-band

information is not available for the 10- and 12-telocentric specimens

collected in the range of N alabamensis, our tentative conclusion is that

N alabamensis is a chromosomally polymorphic species, possibly exhib-

iting clinal variation in number of telocentrics. Resolution of this prob-

ably complex problem awaits more extensive karyological and biochem-

ical investigations.

Necturus maculosus may be the only totally allopatric species of

Necturus, except in regions where it may have been recently introduced

(Ashton et al. 1980), and has by far the largest range of any of the

species. Yet, virtually no molecular divergence is detectable between

populations of this species (Ashton et al. 1980). Specimens of N. punc-

tatus taken from two different river systems less than 200 km apart in

North Carolina showed more genetic divergence from each other than

did specimens of N. maculosus collected from widely disparate localities

in Wisconsin, Massachusetts, and North Carolina (Ashton et al 1980).

The electrophoretic and karyological patterns observed probably reflect

the streambound life style of these neotenic salamanders, which repres-

ent a lineage that may have been permanently aquatic since the Paleo-

cene (Naylor 1978). The isolation of populations of Necturus in parallel

river systems imposes a constraint on dispersal patterns,, and has proba-

bly encouraged in situ chromosomal and genetic differentiation. The

peculiar geographic distribution and pattern of genetic differentiation of

N. maculosus relative to its congeners is probably due to its occupancy

of the vast, highly branched, north-south flowing Mississippi River

system.

The differences observed in the heteromorphic sex chromosomes

among the species of Necturus may reflect evolutionary differentiation

of these elements in a manner similar to that hypothesized by Ohno

(1967). If so, then the sex chromosomes of Necturus species can be used,

in conjunction with electrophoretic and distributional data, to recon-

struct certain aspects of the phylogenetic history of Necturus in North

America.

From a karyological viewpoint, the geographic distribution of Nec-

turus species can be interpreted as a "karyomorphocline", with the south-

east coastal species showing increasing karyological differentiation south-

ward and then westward along the Gulf coast, and finally into the Mis-

sissippi River drainage system (Fig. 6). The Appalachian Mountains

form a natural barrier to westward dispersal of the northern coastal

populations and to eastward dispersal of N maculosus. A somewhat

analogous situation exists in subspecies of the pickerel, Esox america-

nus (Crossman 1966). In contrast to our interpretation of the Necturus

50 Stanley K. Sessions and John E. Wiley

pattern, two subspecies of pickerels are thought to have spread north-

ward, one on each side of the Appalachians from a common origin at

the southern end, with secondary contact at the southern end producing

intergrades (Crossman 1966). Of relevance to the present distribution of

Necturus species as well as Esox americanus, however, is the contrast

between the relatively short, eastward and parallel flowing river systems

on the east side of the Appalachians and the vast Mississippi River sys-

tem flowing southward from Canada to Louisiana on the west side:

north-south spreading of such stream-bound, permanently aquatic organ-

isms was probably very slow along the Atlantic coast relative to south-

north spreading in the inland area.

Presumably, karyological differentiation in Necturus, involving increas-

ing chromosomal asymmetry and progressive changes in sex chromo-

some morphology, gradually occurred as populations became estab-

lished farther south, around the southern end of the Appalachians, and

into the Mississippi River drainage system. Necturus maculosus (includ-

ing the southern form, N beyeri) is the culmination of these karyologi-

cal and geographic trends and represents the most derived state. It has

an extensive, fanlike distribution and probably represents one vast,

genetically and karyologically homogeneous population. The distribu-

tion of N maculosus was probably the result of a relatively recent and

explosive northward dispersal of this species in response to access to the

extensive Mississippi River drainage system. This hypothesis awaits

substantiation by further electrophoretic, karyological, and ecological

studies.

ACKNOWLEDGMENTS.— We are indebted to Ray E. Ashton, Jr.,

William Birkhead, Paul Moler, Richard Sage, K. Thomas, and Wayne

Van Devender for providing the specimens used in this study. The fol-

lowing people read the manuscript and offered valuable comments at

various stages of its preparation: Ray E. Ashton, Jr., Alvin Braswell,

Steven D. Busack, John E. Cooper, James Kezer, James Patton, Steven

Sherwood, and David B. Wake. This research was supported in part by

NSF grant DEB 78-03008.

LITERATURE CITED

Ashton, Ray E., Jr., A.L. Braswell and S.I. Guttman. 1980. Electrophoretic

analysis of three species of Necturus (Amphibia: Proteidae), and the taxo-

nomic status of Necturus lewisi (Brimley). Brimleyana 4:43-46.

Brame, Arden H., Jr. 1967. A list of the world's recent and fossil salamanders.

Herpeton. J. Southwest. Herpetol. Soc. 2(1): 1-26.

Necturus Chromosome Evolution 51

Brandon, Ronald A. 1969. Taxonomic relationship of the salamander genera

Proteus and Necturus. Bull. Natl. Speleol. Soc. 57:33-36.

Brode, W.E. 1970. A systematic study of salamanders of the genus Necturus

(Rafinesque). PhD dissert., Univ. Southern Mississippi, Hattiesburg. 146

pp. Diss. Abstr. Int. B Sci. Eng. 50(1 1): 5288B-5289B.

Conant, Roger. 1975. A Field Guide to Reptiles and Amphibians of Eastern and

Central North America. 2nd ed. Houghton Mifflin Co., Boston. 429 pp.

Crossman, E.J. 1966. A taxonomic study of Esox americanus and its subspecies

in eastern North America. Copeia 1966(1): 1-20.

Gorham, Stanley W. 1974. Checklist of World Amphibians. New Brunswick

Museum, Lingley Printing Co. Ltd., Saint John, NB. 173 pp.

Gould, Stephen J. 1977. Ontogeny and Phylogeny. Belknap Press, Cambridge

and London. 501 pp.

Green, David M., J. P. Bogart and E.H. Anthony. 1980. An interactive,

microcomputer-based karyotype analysis system for phylogenetic cytotax-

onomy. Comput. Biol. Med. 70:219-227.

Hecht, Max K. 1957. A case of parallel evolution in salamanders. Proc. Zool.

Soc. (Calcutta), Mookerjee Mem. Vol.:283-292.

1958. A synopsis of the mudpuppies of eastern North America. Proc.

Staten Island Inst. Arts Sci. 27:3-38.

, and J.L. Edwards. 1976. The determination of parallel or mono-

phyletic relationships: the proteid salamanders — a test case. Am. Nat.

770:653-677.

Kezer, James, and S.K. Sessions. 1979. Chromosome variation in the pletho-

dontid salamander, Aneides ferreus . Chromosoma (Berl.) 77:65-80.

, T. Seto and CM. Pomerat. 1965. Cytological evidence against

parallel evolution of Necturus and Proteus. Am. Nat. 99:153-158.

Larsen, John H., Jr., and D.J. Guthrie. 1974. Parallelism in the Proteidae

reconsidered. Copeia 1974(3):635-643.

Levan, Albert, D. Fredga and A. A. Sandberg. 1964. Nomenclature for centro-

meric position on chromosomes. Hereditas (Lund) 52:201-220.

Morescalchi, Alessandro. 1975. Chromosome evolution in the caudate amphib-

ia. Pp. 339-387 in T. Dobzhansky, M.K. Hecht, W.C. Steere (eds.). Evo-

lutionary Biology, Vol. 8. Plenum Press, New York. 396 pp.

Naylor, Bruce G. 1978. The earliest known Necturus (Amphibia, Uro-

dela), from the Paleocene Ravenscrag Formation of Saskatchewan. J.

Herpetol. 72:565-569.

Noble, G.K. 1931. The Biology of the Amphibia. 1954 reprint, Dover, New

York. 577 pp.

Ohno, Susumu. 1967. Sex Chromosomes and Sex-linked Genes. Monogr.

Endocrinol. I. Springer- Verlag, Berlin-Heidelberg-New York. 192 pp.

Schmid, M., J. Olert and Ch. Klett. 1979. Chromosome banding in amphibia.

III. Sex chromosomes in Triturus. Chromosoma (Berl.) 77:1-14.

Sessions, Stanley K. 1980. Evidence for a highly differentiated sex chromosome

heteromorphism in the salamander, Necturus maculosus (Rafinesque).

Chromosoma (Berl.) 77:157-168.

52 Stanley K. Sessions and John E. Wiley

Seto, Takeshi, CM. Pomerat and J. Kezer. 1964. The chromosomes of Nectu-

rus maculosus as revealed in cultures of leukocytes. Am. Nat. 95:71-78.

White, M.J.D. 1973. Animal Cytology and Evolution. 3d ed. Cambridge Univ.

Press, Cambridge. 961 pp.

Accepted 4 June 1982

The Testis and Reproduction in Male Necturus,

with Emphasis on N. lewisi (Brimley)

Jeffrey pudney

Department of Biology, Boston University

Jacob A. Canick

Brown University,

Division of Biology and Medicine

Department of Pathology

Women 's and Infants ' Hospital

Providence, Rhode Island 02908

AND

Gloria V. Callard

Department of Biology

Boston University, Boston, Massachusetts 02215

ABSTRACT. — Although it has long been recognized that estrogens

are produced by the testis of many vertebrate species the intratesticular

site of aromatization is still controversial. Both interstitial Leydig and

intertubular Sertoli cells have been implicated as the source of testicu-

lar estrogen. The mammalian testis is histologically complex with

seminiferous tubules uniformly distributed amongst the interstitial

tissue throughout the testis, which makes it difficult to localize steroid-

ogenic enzymes within the testis. Urodele amphibians, however, at the

close of the breeding season develop a specialized region of the testis

called the glandular tissue that is essentially composed of Leydig cells

and is formed by hypertrophy of the interlobular Leydig cells following

spermiation of the seminiferous lobules. This glandular tissue in Nec-

turus testis can be visualized with a dissecting microscope and separ-

ated from the seminiferous lobules, an anatomical arrangement that

provides an opportunity to investigate the intratesticular location of

steroidogenic enzymes. In a previous study it was shown that aroma-

tase was localized to the glandular tissue in Necturus maculosus testis.

Thus in N. maculosus Leydig cells are responsible for the production

of testicular estrogens. The testis of Necturus also undergoes a longi-

tudinal wave of spermatogenesis. Due to this topographical

arrangement it is possible by dissection of the testis to obtain regions

with Leydig cells at different stages of differentiation. This was carried

out in N. lewisi towards the end of the breeding season at which time

the testis was divided into cephalic and caudal regions. The cephalic

region contained seminiferous lobules filled with germ cells and undif-

ferentiated interlobular Leydig cells. The caudal region contained

lobules that had undergone spermiation and the Leydig cells had

hypertrophied to form the glandular tissue. Microsomes were prepared

from these two regions and key enzymes for estrogen synthesis, 17a-

hydroxylase and aromatase were measured. Cytochrome P-450, the

catalytic component of these enzymes, was also measured in each

Brimleyana No. 10:53-74. February 1985. 53

54 Jeffrey Pudney, Jacob A. Canick, Gloria V. Callard

region. The results showed that levels of 17a-hydroxylase and aroma-

tase as well as P-450 concentration increased as the glandular tissue

developed. As yet the functional significance of estrogen production by

Necturus testis has not been investigated.

INTRODUCTION

Urodele amphibians of the genus Necturus are widely distributed

throughout the eastern and middle regions of North America, where

they are extremely abundant in rivers tributary to the Great Lakes and

to inland streams and small lakes. The ability to survive in waters of

such diverse characteristics apparently accounts for their wide distri-

bution. Necturus, one of the largest of salamanders, is primarily noctur-

nal, although in very muddy or reedy habitats it may be more or less

active during the day, and is preeminently aquatic. These salamanders

are also usually active throughout the whole year and do not, it seems,

undergo periods of true hibernation.

The sexes of Necturus are similar in both form and coloration. Fol-

lowing a primitive courtship behavior the animals mate, depending on

their regional distribution, throughout the summer and fall. Fertiliza-

tion is internal by means of spermatophores produced by the males. The

male vent is larger than that of the female, becomes inflamed during the

breeding season, and is capable of eversion to expose two papillae that

possibly aid in the deposition of spermatophores into the cloaca of the

female. Cloacal glands occur in the male and are presumably involved

in the formation of spermatophores. The spermatozoa are stored over

the winter months in spermathecae of the females, which then undergo a

short spawning season in the following spring and deposit eggs in rudi-

mentary nests that can be guarded by either parent.

MORPHOLOGY OF NECTURUS TESTIS

As in all amphibia, the paired elongate testes of Necturus are

abdominal in position, bordering the kidneys and attached to the dorsal

body wall by a mesorchium (Fig. 1). Within this mesenteric membrane

lies the vascular system of the testis and vasa efferentia, which convey

the spermatozoa to the Wolffian ducts — long, coiled structures in

which spermatozoa are stored prior to encapsulation within the spermat-

ophores.

The structural unit of Necturus testes is the cyst which contains the

germ cells and associated somatic cells, analogous to Sertoli cells found

in amniote testes. These units are enclosed and contained in larger struc-

tures, the seminiferous lobules. Surrounding the lobules is the interlobu-

lar tissue in which is found the Leydig cells. The organization of the

testis consists of seminiferous lobules radiating outward to the periphery

Necturus Testis and Reproduction

■ m

55

Fig. 1. Gross dissection of Necturus maculosus displaying testes (large arrows)

attached to the dorsal body wall by a mesorchium (small arrows) through which

run the vasa efferentia that empty into the Wolffian duct (arrowheads). From

Pudneyetal. (1983). X 1.5.

of the testis from a main, central, longitudinal collecting duct to which

they are joined by short tubes devoid of germ cells (see Fig. 3). The

main duct, in turn, is connected to the vasa efferentia, thus allowing

egress of spermatozoa from the testis.

In Necturus, as in most urodeles, there is during the breeding cycle

a caudocephalic wave of spermatogenesis along the length of the testis,

56 Jeffrey Pudney, Jacob A. Canick, Gloria V. Callard

resulting in a spatial and temporal segregation of differentiating germ

cells. Depending on the region and presumably regulated by the temper-

ature of the environment, for Necturus this wave of spermatogenesis can

begin in the spring or early summer and is concluded by late summer or

fall. In many urodeles this wave of spermatogenesis is slow. Due to the

tardy differentiation of germ cells in these species the testis becomes

divided into lobes that contain germinal elements at different stages of

development. In Necturus, however, the spermatogenic wave is compar-

atively rapid and thus lobation of the testis does not occur. Further-

more, the process of spermatogenesis is restricted to those regions of the

seminiferous lobules distal to the central collecting duct, while lobules

proximal to this duct contain groups of undifferentiated germ cells, the

spermatogonia (see Fig. 2). These germ cells are in a "resting stage,"

providing a reservoir for successive waves of spermatogenesis. Thus, in

N. lewisi there is a maturational wave of germ cells occurring longitudi-

nally in a caudocephalic direction, plus a proximal to distal cycle of

differentiating germ cells to form the maturing segments of the seminif-

erous lobules that initiate seasonal recrudescence (Fig. 2). Cyst devel-

opment begins when a single large cell, the primary spermatogonium,

becomes associated with several somatic cells. Mitotic divisions of these

primary spermatogonia results in the formation of cysts (enclosed by the

seminiferous lobule), containing secondary spermatogonia. Subsequent

maturational divisons of these spermatogonia cause an increase in the

size of the cysts by the development of spermatocytes that differentiate

into spermatids. During spermiogenesis the developing spermatozoa are

still enclosed by somatic cells and the integrity of the cysts remains

intact. By the time the final stages of spermatozoan maturation are

reached, however, the cysts are highly distended. Presumably due to this

large increase in size the cysts now rupture, resulting in the spermatozoa

(embedded in somatic cells) lying free within the confines of the seminif-

erous lobule. The release of spermatozoa from the somatic cells, by the

act of spermiation, now allows exit of the spermatozoa via the lumen of

the lobules into the main collecting duct. It should be emphasized that

once the cysts enter into the spermatogenetic process the germ cells

present in each of these structures undergo the various developmental

changes synchronously in a given seminiferous lobule. Thus, each lobule

will, in successive periods, contain in its maturing portion, spermato-

cytes, spermatids, and finally spermatozoa. The maturing portion will

never at any one time contain all, or a combination of, these stages as

occurs in the seminiferous tubules of the amniotes.

It has been previously demonstrated that in Necturus maculosus

the longitudinal wave of spermatogenesis is also reflected in the degree

of development of the adjacent interlobular tissue (Humphrey 1921;

Pudney et al. 1983). The same relationship also prevails in the testis of

Necturus Testis and Reproduction

57

N. lewisi. The interlobular cells associated with the immature region of

seminiferous lobules, or those surrounding lobules containing develop-

ing germ cells appear, morphologically, to be undifferentiated (see Fig.

2). When, however, the lobules undergo spermiation this, in as yet an

maturing lobules

• medial zone

caudal zone

ANNUAL

REGROWTH

tunic

• all zones

Fig. 2. Diagrammatic representation of N. maculosus testis towards the close of

spermatogenetic activity. Seminiferous lobules (clear areas) containing the germ-

inal elements empty into the main, central, longitudinal collecting duct. Lobular

portions adjacent (proximal) to this duct contain immature germ cells that act

as a reservoir for successive bouts of spermatogenetic activity. Mature germ cell

cysts are in lobular portions distal to the collecting duct. Lobules are sur-

rounded by an interlobular tissue (grey areas), development of which depends

upon the state of differentiation of the lobules. The kinetics of spermatogenesis

are indicated by arrows showing the proximal to distal development of germ

cells (annual regrowth) and the caudocephalic differentiation of germ cells that

occurs in distal portions of the lobules and their associated interlobular tissue

during the annual breeding season (maturational wave). From Pudney et al.

(1983).

58

Jeffrey Pudney, Jacob A. Canick, Gloria V. Callard

* If ^ *

Fig. 3. Section of testis of Af. fewisi showing seminiferous lobules containing

immature germ cell cysts composed of spermatogonia (large arrow) and somatic

cells (small arrows). Lobules are connected by short ducts, devoid of germ cells,

to the main collecting duct (large arrowhead). Interlobular tissue (small arrow-

heads) is undeveloped. X 350.

Necturus Testis and Reproduction 59

unknown manner, signals the hypertrophy of the interlobular cells sur-

rounding this region. With the subsequent degeneration of the distal

lobular portion, following the release of the spermatozoa, the interlobu-

lar cells continue to differentiate and eventually form what has been

termed the glandular tissue (see Fig. 2). This region was probably first

described in urodeles by Perez (1906), but the actual term glandular

tissue was first used by Champy (1913). Champy was so struck by the

change in color of the testes associated with the formation of the glan-

dular tissue, which is orange/ yellow due to the enormous lipid content

of the interlobular cells, that he likened its development to the corpus

luteum of the mammalian ovary, calling it a "veritable corps jaune

testiculaire."

The development of the glandular tissue was described in Necturus

in a very extensive and comprehensive review by Humphrey (1921). This

study established that the glandular tissue was formed at the completion

of spermatogenesis and was composed, essentially, of hypertrophied

Leydig cells and remnants of degenerating lobules remaining at the close

of the breeding season. Since the Leydig cells only differentiate as the

lobules undergo spermiation, the evolution of the glandular tissue also

proceeds in a caudocephalic direction, following, as it were, in the wake

of spermatogenesis.

Observations of N. lewisi testis towards the close of the breeding

season demonstrate the simultaneous occurrence of discrete regions dis-

playing different stages of germ cell development. Along the entire

length of the testes the lobules adjacent to the collecting duct consist of

short segments containing spermatogonial cysts (Fig. 3). It should be

mentioned that these immature segments are much longer in N. maculo-

sus at the same stage of testicular development. This possibly reflects a

more extensive period of spermatogenesis in N. lewisi, resulting in the

depletion of a greater number of spermatogonial cysts, which in turn

suggests a more extended breeding period for this species than N. macu-

losus. At the end of the breeding season the immature lobular segments

are mostly in a "resting" condition, although a number of mitotic fig-

ures were apparent in this region (Fig. 4). Thus, it seems that initial

germ cell replenishment can occur, albeit at a slow rate, long before the

main wave of spermatogenetic activity takes place. As has also been

demonstrated in N. maculosus (Pudney et al. 1983), in N. lewisi testis

the peripheral portions of the lobule undergo a marked longitudinal

variation in development. Progressing from the anterior to the posterior

end of the testis are seen: 1) highly distended lobules filled with bun-

dles of spermatozoa embedded in somatic cell cytoplasm (Fig. 5); 2)

recently emptied lobules containing residual spermatozoa embedded in

somatic cell remnants (Fig. 6); 3) lobules in a complete state of col-

lapse and regression (Fig. 7). These zones have no definite boundaries;

60

Jeffrey Pudney, Jacob A. Canick, Gloria V. Callard

they merge gradually, forming a continuum of intermediate stages (see

Fig. 5).

In the cephalic zone the lobules contain a patent lumen throughout

their length for the egress of released spermatozoa. These spermatozoa

enter the Wolffian ducts where they are stored prior to spermatophore

formation (Fig. 9). In the central area of the testis the lobules that have

undergone spermiation are often still connected to the immaure por-

tions, but at this stage are usually in the process of being pinched off

from this part of the lobule (see Fig. 6). Finally, in the caudal zone the

degenerating lobular portions become completely separated from the

immature regions and reduced in volume owing to the dissolution of the

somatic cells by fatty degeneration (see Fig. 7).

The interlobular tissue surrounding these stages of lobular devel-

opment also display dramatic differences in differentiation. Observed by

light microscopy, the interlobular cells associated with immature regions

of the lobule resemble fibroblasts in their gross morphology. Observed

by electron microscropy, however, they have been identified in N. macu-

losus as poorly differentiated Leydig cells (Callard et al. 1980). In the

cephalic zone the distended, sperm-filled lobules occupy almost the

entire volume of this region. This made it difficult to locate and identify

the interlobular tissue. When this tissue was studied in N. maculosus by

means of electron microscopy, however, it could be seen to be com-

dz$i>* \ \

Fig. 4. Mitotic figures occasionally occur in immature cysts. X 357.

Necturus Testis and Reproduction 61

Fig. 5. Cephalic region of testis, containing cysts filled with spermatozoa. Inter-

lobular tissue (arrows) is undeveloped. Note that this region merges with the

recently spermiated area at the top. X 121.

posed of immature Leydig cells and myoid cells (Pudney et al. 1983).

The Leydig cells often abutted directly against the basal lamina of the

seminiferous lobule, but usually were excluded from this area by one or

more long cytoplasmic processess developed by myoid cells. Thus, these

62

Jeffrey Pudney, Jacob A. Canick, Gloria V. Callard

>r*5 % J < {•* ; ^"»r -jf jrf*

Fig. 6. In the spermiated region of the testis the immature lobular portions have

pinched off the mature portions (arrowheads). The mature portions are now

degenerating while the interlobular tissue is developing to form the glandular

tissue. X 70.

Necturus Testis and Reproduction 63

myoid cells resemble, both in appearance and position, those present in

the boundary wall of mammalian seminiferous tubules.

Although the intervening stages of lobular development (i.e.

spermatocytes, spermatids) are no longer present in the testes of animals

at this time, it would appear that development of the interlobular cells is

arrested through all stages in which germ cells are present in the lobule.

Following spermiation, however, the interlobular cells now become less

elongated with rounded nuclei. Electron microscope examination of

these cells in N. maculosus has demonstrated that they now possess

abundant organelles normally associated with active steroidogenesis

(Pudney et al. 1983). The regressing portions of the lobules eventually

become surrounded by fully differentiated Leydig cells, which in cross

section form what has been termed the "epithelioid ring" (see Fig. 9)

(Humphrey 1921). This region, which occupies the peripheral part of the

testis, between the terminal segments of the immature lobules and the

testicular capsule, constitutes the newly formed glandular tissue. In the

caudal zone of the testis this finally becomes the glandular tissue proper,

which is markedly increased in volume by further hypertrophy of the

Leydig cells and complete regression of the lobular remnants. The glan-

dular tissue also becomes highly vascular with groups of Leydig cells

closely associated with numerous blood vessels.

Fig. 7. Glandular tissue proper is composed of hypertrophied Leydig cells

(arrowheads) and degenerating remnants of seminiferous lobules (arrows). X 125.

64

Jeffrey Pudney, Jacob A. Canick, Gloria V. Callard

Fig. 8. Wolffian ducts (attached to the mesonephros) filled with spermatozoa.

X 101.

Necturus Testis and Reproduction 65

* 1

W

Fig. 9. The "epithelioid ring" of hypertrophied Leydig cells (arrows) surround-

ing the degenerating lobules (arrowheads). X 330.

STEROID PRODUCTION BY NECTURUS TESTIS

We became interested in Necturus when it was demonstrated that

the testes of N. maculosus possessed exceptionally high aromatase activ-

ity when homogenates of this tissue were incubated with a steroid pre-

cursor [3H]-androstenedione (Callard et al. 1978). Although it has long

been recognized that estrogens can be synthesized and secreted by the

testis of many species, the exact intratesticualr site of aromatization is

still controversial. Thus, both interstitial Leydig cells and intertubular

Sertoli cells have been implicated as the source of testicular estrogens.

Since the previous study had demonstrated such high aromatase activity

in the testes of Necturus, we thought they would be convenient animal

models to study the specific site of estrogen production.

The formation of the peripheral glandular tissue at the end of the

breeding season results in a distinct zonation of Necturus testis, which

can be readily visualized with the aid of a dissecting microscope (Cal-

lard et al. 1980). Hence, the testes can be easily dissected into the glan-

dular tissue, which is composed mostly of fully differentiated Leydig

cells and another component comprised of seminiferous lobules contain-

ing immature germ cells and their associated somatic cells. This fortui-

66 Jeffrey Pudney, Jacob A. Canick, Gloria V. Callard

tous arrangement thus allows a direct comparison to be made of the

ability of these isolated tissue compartments to formulate estrogens and

so provide information on the exact site of aromatization in Necturus

testes. A recent study using these isolated tissues from N. maculosus

testes demonstrated that the glandular tissue contained two key enzymes

in estrogen biosynthesis, 17 a-hydroxylase and aromatase (Callard et al.

1980). Also, spectral measurements showed that cytochrome P-450 spe-

cies that bind progesterone and androstenedione, respectively, the ster-

oidal substrates for 17 a-hydroxylase and aromatase, were concentrated

in the glandular tissue. Levels of both the steroidogenic enzymes and

cytochrome P-450 were negligible in the isolated seminiferous lobule

fractions, indicating that the glandular tissue and its Leydig cells (identi-

fied by electon microscope observations) were the site of aromatization

in the testes of Necturus. This study on N. maculosus was carried out in

the fall when the glandular tissue was fully developed along the entire

length of the testes.

The longitudinal wave of spermatogenesis that Necturus undergoes

results in the spatial segregation of both germ cells and Leydig cells at

different stages of development in the testes during the breeding season.

This is an important consideration, since the relationship between sper-

matogenesis and Leydig cells during specific stages of germ cell devel-

opment is very difficult to study in most common laboratory animals.

In these species all spermatogenic stages occur simultaneously in the

testes from the onset of puberty. Moreover, the seminiferous tubules

and interstitial tissues are uniformly distributed and intermingled through-

out the entire organ. Therefore, using these species it is technically diffi-

cult to obtain precise information on the functional interdependence of

the two tissue compartments without disrupting their normal anatomi-

cal relationships. These difficulties, however, can be circumvented by

studying the testes of Necturus with its discrete temporal and spatial

segregation of germ cell stages and accompanying interlobular tissue

(Pudney at al. 1983).

Figure 2 demonstrates salient morphological features that have

been diagrammatically represented for the testis of N. maculosus as it

appears towards the close of the breeding season (Pudney et al. 1983).

At this time of the year essentially the same anatomical arrangment of

tissues also occurs in the testes of N. lewisi, which were separated trans-

versely into a cephalic region and a caudal region. Both regions con-

tained the immature portions of the seminiferous lobules and associated

undifferentiated interlobular tissue. The cephalic region, however, also

possessed the maturing lobular portions filled with cysts containing

spermatozoa, while the caudal region was composed of the spermiated

degenerating lobules plus the hypertrophied Leydig cells forming the

glandular tissue. The isolated regions were analyzed for 17 a-hydrox-

Necturus Testis and Reproduction 67

ylase and aromatase activity and cytochrome P-450 content. These func-

tional parameters were then correlated, by means of electron micros-

copy, with the morphology of the Leydig cells present in the two

regions. In this way a direct comparison could be made concerning the

morphological appearance of Leydig cells, associated with different

stages of the spermatogenetic cycle, and their capacity for aromatization.

The results of the assays for 17 a-hydroxylase and aromatase in

microsomes prepared from the different regions of N. lewisi testes are

summarized in Table I. Under our assay conditions, 17 a-hydroxy-

progesterone is the sole product synthesized from the substrate, [3H]-

progesterone. In all experiments both estrone and estradiol- 17 f3 are

products of aromatization using either [3H] 19-Hydroxyandrostenedione

or [3H] androstenedione as substrates for this enzyme. Previously we

localized these steroidogenic enzymes in fully developed glandular tissue

comprised of highly differentiated Leydig cells (Callard et al. 1980). In

N. lewisi, however, due to the temporal formation of the glandular

tissue, we have been able to show that the activity of these enzymes is

related to the degree of differentiation of the Leydig cells. Similar

results have been demonstrated in the testis of N. maculosus at the same

stage of the breeding cycle (Pudney et al. 1983). Thus, in both species

the enzymes increased in activity progressively from the least mature

anterior segment of the testis, containing poorly developed Leydig cells,

to the posterior segment possessing the glandular tissue comprised of

fully differentiated Leydig cells. Cytochrome P-450, which is the cata-

lytic component of each of the steroidogenic enzymes studied here, was

also measured in microsomes prepared from each of the testicular seg-

ments. Differences in cytochrome P-450 concentration in these regions

was also found to closely reflect the observed changes in activity of

these enzymes (Table I).

DISCUSSION

In his study of N. maculosus, Humphrey (1921) stated that the

glandular tissue developed not only by hypertrophy of the Leydig cells,

but also by mitotic activity of these cells. The present study on N. lewisi

(and also observations on N. maculosus) did not demonstrate mitotic

figures in the glandular tissue, at any state in its formation. These con-

flicting observations are difficult to reconcile, unless the mitoses of the

differentiating Leydig cells are so transient they can only be detected

during extremely short periods of the breeding season, not examined in

our investigations.

It would be pertinent to discuss briefly, at this stage, the derivation

of the interlobular Leydig cells present in the testis of N. lewisi. This is

an important issue since it has long been accepted in some anamniote

classes, such as teleosts and urodele amphibians, that definitive Leydig

68

Jeffrey Pudney, Jacob A. Canick, Gloria V. Callard

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70 Jeffrey Pudney, Jacob A. Canick, Gloria V. Callard

cells are not actually present in the interlobular tissue but occur as a

component of the boundary wall surrounding the seminiferous lobules.

This concept of Leydig cell development was originated by Marshall

and Lofts (1956) who suggested, from investigations restricted to light

microscopic observations of frozen sections colored with a Sudan dye,

that in the teleost testis a lobular boundary cell was the homologue of

the Leydig cell present in the testis of these amniotes. This initial observ-

ation eventually became expanded to include the urodele amphibia

where it has been routinely reported that lobular boundary, perilobular,

or pericystic cells were analogous, if not homologous, to Leydig cells of

the amniote testis (see review by Lofts 1968; Roosen-Runge 1977; Pils-

worth and Setchell 1981). This concept, however, became confused

when, from various descriptions of the teleost testis using the more criti-

cal resolving power of the electron microscope, it was reported that the

lobular boundary cell actually corresponds to the Sertoli cell (see review

by Grier 1981). The situation is further complicated in that anamniotes

undergo a seasonal cycle of testicular activity, and so, depending upon

the stage of spermatogenesis, Leydig cells may or may not appear con-

spicuous. This becomes an important point if the observations being

carried out use the light microscope for examination of testis sections,

since the resolution of this instrument often precludes the positive loca-

tion and identity of Leydig cells. An attempt to clarify the question

concerning the presence and identity of Leydig cells in anamniotes has

recently been carried out by Grier (1981).

Grier (1981) re-evaluated the literature pertaining to the structure

of the teleost testis and concluded that the term lobular boundary cell

was no longer tenable as the definition of Leydig cells in these anamni-

otes. In fact, in this vertebrate group the description of the lobular

boundary cells as Leydig cells is erroneous. From its location, and other

morphological criteria, the lobular boundary cell actually represents the

Sertoli cell of the teleost testis. In view of this, Grier strongly stated that

the term lobular boundary cell used to describe Leydig cells in the tele-

ost testis be discontinued. In our studies on Necturus testis we have

reached a similar conclusion. First, Leydig cells at various stages of dif-

ferentiation appear to be a constant component of Necturus testis

throughout the year. Secondly, these Leydig cells are present in the

interlobular compartment arising from precursor cells that are a per-

manent feature of this tissue. Thus, the concept of the lobular boundary

cell is also no longer applicable in the species that we have investigated.

Furthermore, the presence of myoid cells in the interlobular tissue of N.

maculosus testis, which often excluded the Leydig cells from direct con-

tact with the seminiferous lobules, is morphologically reminiscent of the

anatomical arrangement present in the amniote testis. It would seem,

therefore, that the organization of the testis in Necturus, and possibly in

Necturus Testis and Reproduction 71

all urodele amphibians, is similar to but less complex than that of the

amniote testis.

The biochemical parameters measured in N. lewisi testis, indicating

active steroidogenesis, essentially correspond with the topographical

location and morphological differentiation of the Leydig cells. Thus, it

would appear that these Leydig cells are responsible for the production

of steroids by the testis of N. lewisi. Their steroidogenic potential also

appears to be related to the cycle of the seminiferous epithelium, since

spermiation, followed by regression of the mature lobules, is the event

signaling hypertrophy of the surrounding interlobular tissue. It is possi-

ble that similar changes also occur in mammals but have never been

detected, since not only are different germ cell stages normally found

throughout the testis year-round in most common laboratory animals

but differentiation is not synchronized in all germ cells of any one tes-

ticular region. Recent studies have in fact shown that when Leydig cells

from adult rats are separated on density gradients, at least three func-

tionally distinct populations can be identified (O'Shaugnessy et al.

1981). Whether these Leydig cells are randomly distributed throughout

the testis of the rat or actually occur in specific relation to certain stages

of germ cell development, however, remains to be elucidated. An intrig-

uing question is the mechanism by which the spermiated, degenerating

lobules of Necturus testis initiate the hypertrophy and differentiation of

the Leydig cells associated with these regions. This seems to be a gener-

alized phenomenon, since there are scattered reports in the literature

demonstrating, in many mammalian species, that damage to the semi-

niferous epithelium, either by physical or chemical agents, results in the

hypertrophy of Leydig cells adjacent to these lesions (Neaves 1975; Aoki

and Fawcett 1978). Furthermore, inhibition of spermatogenesis due to

cryptorchidism is similarly associated with an increase in development

of Leydig cells as well as an enhancement of androgen secretion (deK-

retser et al. 1980). As yet, however, further investigations are required to

determine the molecular events controlling this interesting relationship

between the spermatogenic tissue and Leydig cells.

It has been assumed that, in all species, at least some stages of

spermatogenesis are androgen-dependent (Callard, I. P. et al. 1978;

Rodriguez-Rigau et al. 1980). In Necturus testis, therefore, it seems

paradoxical that lobular regions which contain differentiating germ cells

are found to be associated with poorly developed Leydig cells possessing

low androgen synthesizing abilities. It is possible, however, that these

Leydig cells do synthesize low but adequate quantities of androgens that

suffice for local spermatogenetic requirements. Among non-mammals it

has been commonly demonstrated that spermatogenetic activity and

Leydig cell development are often temporally separated during the

annual cycle (Lofts and Bern 1972; Callard, I. P. et al. 1978). Where

72 Jeffrey Pudney, Jacob A. Canick, Gloria V. Callard

measurements have been made, it has been found in some species that,

although intratesticular androgen levels are high during spermatogene-

sis, the rise in plasma androgen and the display of breeding behavior

corresponds to the time of maximal Leydig cell hypertrophy (Courty

and Dufaure 1979). These observations, therefore, lead us to the possi-

bility that the development of a large volume of Leydig tissue, synthesiz-

ing high levels of androgen and estrogen, as for example the glandular

tissue of N. lewisi, may be an adaptation for the secretion of steroids

into the circulation (see Pudney et al., 1983). The extensive vasculariza-

tion of the glandular tissue and the timing of its development following

spermiation tends to support this view. The function of these steroids

produced by the glandular tissue is unknown, although exceptionally

high levels of circulating androgen and estrogen have been measured in

N. maculosus (Bolaffi and Callard 1979, 1981). Presumably they stimu-

late and maintain development of the secondary sex organs such as the

Wolffian ducts, in which spermatozoa are stored prior to mating, as

well as the cloacal glands and other spermatophore producing structural

paraphernalia. Furthermore, the role any of these steroids plays in the

control of behavioral patterns and nuptial coloration resulting in the

successful breeding of Necturus can be inferred. Of interest in this

respect are reports demonstrating that estrogens in the boar have been

shown to be important in conditioning sexual behavior (Joshi and Rae-

side 1973).

The extraordinarily high rate of estrogen production and its func-

tion within the testis of Necturus remains, however, obscure. Although

some of this estrogen probably affects other body tissues, the presence

of receptors in the testis of Necturus maculosus (Mak et al. 1983) indi-

cates an action in situ as well. It seems reasonable to predict that estro-

gens may exert a direct inhibitory effect on Leydig cell steroidogenesis

and thus signal the demise of the glandular tissue, especially since

aromatase activity is maximal towards the end of the breeding cycle. A

remarkable parallel occurs in the primate corpus luteum, another highly

developed glandular tissue in which degeneration (luteolysis) is initiated

by direct application of estrogen (Karsch and Sutton 1976). This, inter-

estingly enough, brings us back to the original observation by Champy

(1913) who suggested that the glandular tissue developed by Necturus

was in fact a "corpus luteum of the testis".

The above account of our studies on Necturus testis raises and

leaves unanswered many interesting and intriguing questions, both at

the morphological and biochemical levels. This is the way of research.

In seeking an understanding of nature's mysteries, one is usually left

with more imponderables than one started with— which keeps the busi-

ness of research alive. In the field of male reproduction, despite the

efforts of numerous workers past and present, our understanding of the

Necturus Testis and Reproduction 73

mechanisms controlling spermatogenesis is still rudimentary. It would

appear that in order to approach and elucidate some of these problems

more attention should be made in choosing the correct and appropriate

animal model. Nature, fortunately, has been very generous to investiga-

tors of male reproduction by offering a wide range of animals displaying

different reproductive strategies. No one species per se is capable of

providing all the answers. By judicious selection, however, one can elect

to investigate a particular animal because, due to some unique or novel

morphological or biochemical parameter, it is more suited for studying

one particular aspect of male reproduction. Thus, in Necturus testis the

development of the glandular tissue plus the formation of high levels of

estrogen make this species an excellent animal model for studying the

relationship between spermatogenesis and Leydig cells and the role of

estrogen in the testis. Both these problems are difficult to approach in

the more acceptable laboratory animals, which illustrates and defines

the importance of studying the so-called unconventional animal models

in order to understand more fully the phenomena associated with male

reproduction.

ACKNOWLEDGMENTS.— We would like to acknowledge and

thank Ray E. Ashton, Jr., North Carolina State Museum of Natural

History, for supplying specimens of Necturus lewisi. Supported by

USPHS HD16715.

LITERATURE CITED

Aoki, Augustine, and D. W. Fawcett. 1978. Is there a local feedback from the

seminiferous tubules affecting activity of the Leydig cells? Bio. Reprod.

79:144-158.

Bolaffi, Jan L., and I. P. Callard. 1979. Plasma steroid profiles in male and

female mudpuppies Necturus maculosus. Gen. Comp. Endocrinol.

37:443-450.

, and 1981. In vitro regulation of steroidogenesis by ovine

gonadotropins in male and female mud-puppies Necturus maculosus Rafi-

nesque. Gen. Comp. Endocrinol. 44: 108-1 16.

Callard, Gloria V., Z. Petro and K. J. Ryan. 1978. Phylogenetic distribution of

aromatase and other androgen-converting enzymes in the central nervous

system. Endocrinology 705:2283-2290.

, J. A. Canick and J. Pudney. 1980. Estrogen synthesis in Leydig cells:

structural-functional correlations in Necturus testis. Biol. Reprod.

25:461-481.

Callard, Ian P., G. V. Callard, V. Lance, J. L. Bolaffi and J. S. Rossett. 1978.

Testicular regulation in non-mammalian vertebrates. Biol. Reprod. 75:16-43.

Champy, Christian. 1913. De l'existence d'un tissu glandulaire endocrine tem-

poraire dans le testicule (corps jaune testiculaire). C. R. Seances Soc. Biol.

74:367-368.

Courty, Yves, and J. P. Dufaure. 1979. Levels of testosterone in the plasma and

testis of the viviparous lizard (Lacerta vivipara Jacquin) during the annual

cycle. Gen. Comp. Endocrinol. 59:336-342.

74 Jeffrey Pudney, Jacob A. Canick, Gloria V. Callard

de Kretser, David M., J. B. Ker, K. A. Rich, G. Riabridger and M. Dobos.

1980. Hormonal factors involved in normal spermatogenesis and following

the disruption of spermatogenesis. Pp. 107-115 in A. Steinberger and E.

Steinberger (eds.). Testicular Development, Structure and Function. Raven

Press, New York. 536 pp.

Grier, Harry J. 1981. Cellular organization of the testis and spermatogenesis in

fishes. Am. Zool. 27:345-357.

Humphrey, Roger R. 1921. The interstitial cells of the urodele testis. Am. J.

Anat. 29:213-279.

Joshi, Harold S., and J. I. Raeside. 1973. Synergistic effects of testosterone and

oestrogens on accessory sex glands and sexual behavior of the boar. J.

Reprod. Fertil. 35:41 1-423.

Karsch, Fred J., and G. P. Sutton. 1976. An intra-ovarian site for the luteolytic

action of estrogen in the Rhesus monkey. Endocrinology 95:553-561.

Lofts, Brian. 1968. Patterns of testicular activity. Pp. 239-304 in E.J.W. Bar-

rington and C. B. Jorgenson (eds.). Perspectives in Endocrinology, Hor-

mones in the Lives of Lower Vertebrates. Academic Press, London. 523 pp.

, and A. A. Bern. 1972. The functional morphology of steroidogenic

tissues. Pp. 37-126 in D. R. Idler (ed.). Steroids in Non-mammalian Ver-

tebrates. Academic Press, New York. 504 pp.

Mak, Paul, I. P. Callard and G V. Callard. 1983. Characterization of an estro-

gen receptor in the testis of the urodele amphibian Necturus maculosus.

Biol. Reprod. 25:261-270.

Marshall, Alan J., and B. Lofts. 1956. The Leydig-cell homologue in certain

teleost fishes. Nature (Lond.) 777:704-705.

Neaves, William B. 1975. Leydig cells. Contraception 2:571-604.

O'Shaughnessy, Peter J., K. L. Wong and A. H. Payne. 1981. Differential ste-

roidogenic enzyme activities in different populations of rat Leydig cells.

Endocrinology 709:1061-1066.

Perez, Michele C. H. 1906. Resorption phagocytaire des spermatozoides chez les

Tritons. C. R. Seances Soc. Biol. 56:783-784.

Pilsworth, Larry M., and B. P. Setchell. 1981. Spermatogenic and endocrine

functions of the testes of invertebrate and vertebrate animals. Pp. 9-38 in

H. Burger and D. de Kretser (eds.). The Testis. Raven Press, New York.

442 pp.

Pudney, Jeffrey, J. A. Canick, P. Mak and G. V. Callard. 1983. Topographical

distribution of steroidogenic activity in the testis of Necturus. Gen. Comp.

Endocrinol. 50:43-66.

Rodriguez-Rigau, Luis J., Z. Zuckerman, D. B. Weiss, K. D. Smith and E.

Steinberger. 1980. Hormonal control of spermatogenesis in man: compar-

ison with rat. Pp. 139-146 in A. Steinberger and E. Steinberger (eds.).

Testicular Development, Structure and Function. Raven Press, New York.

536 pp.

Roosen-Runge, Edward C. 1977. The Process of Spermatogenesis in Animals.

Cambridge University Press, Cambridge. 214 pp.

Accepted 22 October 1984

Salamander Skin Toxins, With Special Reference To

Necturus lewisi

Ronald A. Brandon

Department of Zoology,

Southern Illinois University at Carbondale,

Carbondale, Illinois 62901

AND

James E. Huheey

Department of Chemistry,

University of Maryland, College Park, Maryland 20742

ABSTRACT. — Crude aqueous extracts of skin from two specimens of

Necturus lewisi were found to cause strong symptoms of toxinosis in

bioassay mice when injected intraperitoneally. The symptoms resembled

those caused by injections of pseudotritontoxin and skin extracts from

a variety of other salamanders, but only one bioassay mouse died. In

comparison to the effects of injected skin extracts of other salamanders

on bioassay mice, the skin of N. lewisi is moderately active in causing

distress but relatively less lethal. Effects of per os administration on

potential natural predators remain to be tested.

INTRODUCTION

The skins of most amphibians — lacking hair, feathers, epidermal

scales, dermal armor, or significant keratinization — are relatively thin,

heavily glandular, covered with a mucoid coat, and serve as the major

physiological interface through which ions, respiratory gases, and water

are exchanged with the environment (Whitear 1977). This sort of moist

integument in an environment rich in bacteria, fungi, and yeasts is par-

ticularly susceptible to invasion by microorganisms and to major physi-

ological disruption if the protective mucoid coat is damaged. In addi-

tion to containing many physiologically active regulatory chemicals, the

skin is bathed in mucus and other glandular products that may contain

a variety of chemical materials — proteins, peptides, steroids, alkaloids,

and simple biogenic amines (Cei et al. 1967, 1968; Daly et al. 1977; Daly

and Heatwole 1966; Erspamer et al. 1962, 1964; Habermehl 1974; Jaussi

and Kunz 1978; Flier et al. 1980; Mensah-Dwumah and Daly 1978;

Endean et al. 1975). Some of these chemicals have an antimicrobial

function (Habermehl 1975; Habermehl and Preusser 1969, 1970; Preusser

et al. 1975). Others may function as stress- warning markers (Hedberg

1981). In many species the chemicals have adaptive significance in rend-

ering the animals noxious or toxic to predators, or provide tastes and

smells that predators associate with noxious or toxic effects (Brandon

Brimleyana No. 10:75-82. February 1985. 75

76 Ronald A. Brandon and James E. Huheey

and Huheey 1981; Brandon et al. 1979a,b; Brodie 1968a,b, 1971, 1977;

Brodie and Gibson 1969; Brodie and Howard 1973; Brodie et al. 1974,

1979; Dodd et al. 1974; Dodd and Brodie 1976; Hensel and Brodie 1976;

Howard and Brodie 1973; Huheey 1960; Hurlbert 1970; Nickerson and

Mays 1973; Pough 1971; and Webster 1960).

Among salamanders, skin toxicity has been examined in detail in

Asiatic, European, and North American salamandrids and to a lesser

degree among plethodontids. Wakeley et al. (1966) found tetrodotoxin

in significant amounts in two species of Cynops, three species of Tari-

cha, and one species of Notophthalmus; trace amounts were found in

four species of Triturus. Brodie et al. (1974) found a species of Parame-

sotriton also to have highly toxic skin, apparently because of tetrodo-

toxin. Wakeley et al. (1966) looked for tetrodotoxin in several non-

salamandrids — Necturus maculosus, Amphiuma sp., Siren lacertina,

Ensatina eschscholtzii, Batrachoseps attenuatus, and Amides lugubris —

but found none. The toxic alkaloids of Salamandra salamandra have

been studied in great detail (Habermehl 1974), and Triturus cristatus

has recently been shown to secrete a toxic protein (Jaussi and Kunz

1978).

Skin extracts containing toxins of high molecular weight have been

obtained from Taricha torosa, T. granulosa, Notophthalmus virides-

cens, and the plethodontids Pseudotriton ruber and P. montanus

(Brandon and Huheey 1981; Huheey and Brandon 1977). They may also

be present in some other plethodontids as well (Brandon and Huheey

1981; Phisalix 1922; Table 1), but skin extracts of most species of sala-

mander remain to be examined. Several other species of plethodontids

are demonstrably of reduced palatability to natural and experimental

predators (Brodie 1977; Brodie and Howard 1973; Brodie et al. 1979;

Dodd and Brodie 1976; Dodd et al. 1974; Huheey 1960). Some species

appear to be noxious but not toxic, but to this time skin extracts of all

species tested except one of Desmognathus and two of Plethodon have

produced toxic symptoms in bioassay animals, and only some species of

Desmognathus and some populations of Plethodon jordani seem com-

pletely palatable to predators.

The objective of this report is to describe the effects of crude skin

extracts of Necturus lewisi on bioassay mice within the context of sim-

ilar tests of extracts from a variety of other species.

MATERIALS AND METHODS

Each of two live specimens of Necturus lewisi was measured (snout-

vent length, mm), weighed (nearest mg), then killed by decapitation and

pithed. All skin was dissected from the trunk between hind and front

limbs, weighed to the nearest mg, and measured to the nearest mm. The

skin was immediately minced and frozen in liquid nitrogen and reduced

Salamander Skin Toxins 77

Table 1. Bioassay results of white mice injected i.p. with crude skin extracts

from two specimens of Necturus lewisi.

Dosage (mg/ kg) Result

to a granular powder with a mortar and pestle. The ground sample was

slurried in 4-5 ml distilled water for 5 minutes to extract the toxins, then

centrifuged for 2 minutes at 3600 rpm, and the supernatant decanted

into a sterile centrifuge tube. The residue was rinsed twice with 1-ml

portions of distilled water and the supernatant decanted, after centrifu-

gation, into the sterile centrifuge tube. The exact volume was recorded

for calculation of dose levels. The sample was kept chilled in ice water

at all times except when centrifuged. For all quantitative studies the

extract was either tested immediately, or frozen in an ultra-refrigerator

(-70 ° C) and lyophilized in a Virtis freezedrier for storage. Lyophilized

samples from other salamanders were kept at -15 °C in the freezing

compartment of a refrigerator and showed no detectable deterioration

over time.

Crude extracts were injected i.p. into bioassay mice (Cox Standard

Outbred) weighing 18-25 g, in mg of sample per kg bodyweight dosages.

Mice were also injected with distilled water, Ringer's solution, and skin

extracts of Desmognathus monticola as controls; none of these caused

symptons. Crude extracts were not toxic enough to yield LD5o data but

were bioassayed in mice as completely as possible until the sample was

expended. Detailed notes were kept of symptoms in bioassay mice to all

dosages of crude skin extract.

RESULTS

Following injection of a lethal i.p. dosage of skin extract into a

mouse, symptoms appeared within a few minutes and consisted of hind

leg stretches (repeated hyperextension of legs and lower back), abdomi-

nal compression, occasional hind leg kicks, and wobbly gaint, followed

by extreme irritability, then quiescence. The irritability was elicited by

contact or impending contact with other mice, a blunt instrument,

78

Ronald A. Brandon and James E. Huheey

sounds, and vibrations. The mice showed a characteristic and distinctive

behavior of attempting to escape the stimulus. Leg stretches, abdominal

compression, leg kicks, wobbly gait, quiescence, and irritability occurred

at all dosages, but most mice seemed gradually to overcome the effects.

Relatively larger dosages caused an increase in symptoms and one

resulted in death after 2 1/2 hours.

The bioassay data are summarized in Table 2. Although all dosages

caused symptoms within the range of 300-12,456 mg/kg, only one

mouse died, at a dosage of 7,756 mg/kg.

Table 2. Bioassay results of white mice injected

various salamanders. Dosage expressed

per kg mouse weight.

Species Dosage (mg/kg)

i.p. with crude skin extracts of

as mg salamander skin injected

Result

N

Salamander Skin Toxins 79

Symptoms caused by i.p. injections of crude N. lewisi skin extract

were quite similar to those caused by extracts of Pseudotriton, those of

several other species of hemidactyline plethodontids, and two species of

Ambystoma, and the high-molecular weight fractions of newt skin

extracts (Brandon and Huheey 1981; Huheey and Brandon 1977; Table

2). We realize these may be generalized symptons of distress, but they

are distinct from those caused by tetrodotoxin toxinosis, and they may

be characteristic of protein toxins.

In the context of symptomology, the skin of N. lewisi is moderately

active, in lethality moderately inactive. Without knowledge of the effects

of N. lewisi skin secretion on natural predators, it is not possible to

interpret its effectiveness in predator avoidance. Both Neill (1963) and

Shoop and Gunning (1967) found that Necturus activity away from

cover may be limited by predatory fishes, suggesting that, at least

against these predators, skin secretions are not noxious. Experiments of

palatability to a variety of potential predators are clearly called for.

Antipredator mechanisms have been studied mainly in terrestrial species

in which the action of noxious and toxic skin secretions has been rein-

forced by defensive postures and, often, aposematic coloration, vocali-

zation, and biting (Brodie 1977, 1978). Antipredator mechanisms of

permanently aquatic species have not been examined in detail, although

some are known to produce noxious and toxic skin secretions (e.g.,

Cryptobranchus alleganiensis , Brodie 1971; Nickerson and Mays 1973;

and Andrias japonicus , pers. observ.).

ACKNOWLEDGMENTS.— This work was supported by NSF grants

BMS74-14371 and DEB78-05959. We are grateful to Anthony Paparo

for the use of his laboratory facilities. Alvin L. Braswell, N.C. State

Museum, kindly made the specimens of Necturus available for study.

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Brandon, Ronald A., and J. E. Huheey. 1981. Toxicity in the plethodontid

salamanders Pseudotriton ruber and Pseudotriton montanus (Amphibia,

Caudata). Toxicon 79:25-31.

, G. M. Labanick and J. E. Huheey. 1979a. Learned avoidance of

brown efts, Notophthalmus viridescens louisianensis (Amphibia, Urodela,

Salamandridae), by chickens. J. Herpetol. 75:171-176.

, and 1979b. Relative palatability, defensive

behavior, and mimetic relationships of red salamanders {Pseudotriton

ruber), mud salamanders {Pseudotriton montanus), and red efts {Notoph-

thalmus viridescens). Herpetologica 55:289-303.

Brodie, Edmund D., Jr. 1968a. Investigations on the skin toxin of the red-

spotted newt, Notophthalmus viridescens viridescens. Am. Midi. Nat.

50:276-280.

80 Ronald A. Brandon and James E. Huheey

.. 1968b. Investigations on the skin toxin of the adult rough-skinned

newt, Taricha granulosa. Copeia 1968(2):307-313.

1971. Two more toxic salamanders: Ambystoma maculatum and

Cryptobranchus alleganiensis . Herpetol. Rev. 5:8.

1977. Salamander antipredator postures. Copeia 1977(3):523-535.

1978. Biting and vocalization as antipredator mechanisms in terres-

trial salamanders. Copeia 1978(1): 127-129.

, and L. S. Gibson. 1969. Defensive behavior and skin glands of the

northwestern salamander, Ambystoma gracile. Herpetologica 25:187-194.

, and R. R. Howard. 1973. Experimental study of Batesian mimicry in

the salamanders Plethodon jordani and Desmognathus ochrophaeus. Am.

Midi. Nat. 90:38-46.

, J. L. Hensel and J. A. Johnson. 1974. Toxicity of the urodele

amphibians Taricha, Notophthalmus, Cynops, and Paramesotriton (Sala-

mandridae). Copeia 1974(2): 506-5 11.

, R. T. Nowak and W. R. Harvey. 1979. The effectiveness of antipre-

dator secretions and behavior of selected salamanders against shrews.

Copeia 1979(2):270-274.

Cei, J. M., V. Erspamer and M. Roseghini. 1967. Taxonomic and evolutionary

significance of biogenic amines and polypeptides occurring in amphibian

skin. I. Neotropical leptodactylid frogs. Syst. Zool. 76:328-342.

, , and 1968. Taxonomic and evolutionary sig-

nificance of biogenic amines and polypeptides in amphibian skin. II. Toads

of the genera Bufo and Melanophryniscus . Syst. Zool. 77:232-245.

Daly, John W., and H. Heatwole. 1966. Occurrence of carnosine in skin of

certain leptodactylid frogs. Experientia 22:764-765.

, G. B. Brown, M. Mensah-Dwumah and C. W. Myers. 1977. Classi-

fication of skin alkaloids from neotropical poision-dart frogs (Dendrobati-

dae). Toxicon 76:163-188.

Dodd, C. Kenneth, Jr., and E. D. Brodie, Jr. 1976. Defensive mechanisms of

neotropical salamanders with an experimental analysis of immobility and

the effect of temperature on immobility. Herpetologica 32:269-290.

, J. A. Johnson and E. D. Brodie, Jr. 1974. Noxious skin secretions of

an eastern small Plethodon, P. nettingi hubrichti. J. Herpetol. 5:89-92.

Endean, R., V. Erspamer, G. Falconieri, G. Improta, P. Melchiorri, L. Negri

and N. Sopranzi. 1975. Parallel bioassay of bombesin and litorin, a

bombesin-like peptide from the skin of Litoria aurea. Br. J. Pharmacol.

55:213-219.

Erspamer, V., G. Bertaccini and J. M. Cei. 1962. Occurrence of bradykinin-like

substances in the amphibian skin. Experientia 75:563-564.

, A. Anastasi, G. Bertaccini and J. M. Cei. 1964. Structure and phar-

macological actions of physalaemin, the main active polypeptide of the skin

of Physalaemus fuscumaculatus . Experientia 20:489-490.

Flier, Jeffrey, M. W. Edwards, J. W. Daly and C. W. Myers. 1980. Widespread

occurrence in frogs and toads of skin compounds interacting with the

oaubain site of Na+, K+-ATPase. Science 205:503-505.

Salamander Skin Toxins 81

Habermehl, Gerhard G. 1974. Venoms of Amphibia. Pp. 161-183 in M. Florkin

and B. T. Scheer (eds.). Chemical Zoology, Vol. IX. Academic Press, New

York. 537 pp.

1975. Die biologische Bedeutung tieruscher Gifte. Naturwissen-

schaften 62:15-21.

, and H.-J. Preusser. 1969. Hemmung des Wachstrums von Pilzen

und Bakterien durch das Hautdrusensekret von Salamandra maculosa. Z.

Naturforsch. Teil B. Anorg. Chem. Org. Chem. 24:1599-1601.

, and 1970. Antimikrobielle Aktivitat von Amphibien-

Hautdrusensekreten. II. Substanzen aus Leptodactylus pentadactylus. Z.

Naturforsch. Teil B. Anorg. Chem. Org. Chem. 25:1451-1452.

Hedberg, Thomas G. 1981. A possible stress-warning marker in ambystomatid

salamanders. J. Exp. Zool. 276:349-355.

Hensel, John L., Jr., and E. D. Brodie, Jr. 1976. An experimental study of

aposematic coloration in the salamander Plethodon jordani. Copeia

1976(l):59-65.

Howard, Ronnie R., and E. D. Brodie, Jr. 1973. A Batesian mimetic complex in

salamanders: responses of avian predators. Herpetologica 29:33-41.

Huheey, James E. 1960. Mimicry in the color pattern of certain Appalachian

salamanders. J. Elisha Mitchell Sci. Soc. 76:246-251.

, and R. A. Brandon. 1977. Novel toxins and the question of warning

coloration and mimicry in salamanders. Herpetol. Rev. 8(3) Suppl.:10.

Hurlbert, Stuart H. 1970. Predator responses to vermilion-spotted newt

(Notophthalmus viridescens). J. Herpetol. 4:47-55.

Jaussi, R., and P. A. Kunz. 1978. Isolation of the major toxic protein from the

skin venom of the crested newt, Triturus cristatus. Experientia 54:503-504.

Mensah-Dwumah, Monica, and J. W. Daly. 1978. Pharmacological activity of

alkaloids from poison-dart frogs (Dendrobatidae). Toxicon 76:189-194.

Neill, Wilfred T. 1963. Notes on the Alabama waterdog, Necturus alabamensis

Viosca. Herpetologica 79:166-174.

Nickerson, Max A., and C. E. Mays. 1973. The hellbenders: North American

"giant salamanders." Milw. Public Mus. Publ. Biol. Geol. No. 1. 106 pp.

Phisalix, Marie. 1922. Animaux venimeux et venins, Vol. 2. Masson, Paris. 864

pp.

Pough, F. Harvey. 1971. Leech-repellent property of eastern red-spotted newts,

Notophthalmus viridescens. Science 774:1144.

Preusser, H.-J., G. Habermehl, M. Sablofski and D. Schmall-Haury. 1975.

Antimicrobial activity of alkaloids from amphibian venoms and effects on

the ultrastructure of yeast cells. Toxicon 73:285-289.

Shoop, C. Robert, and G. E. Gunning. 1967. Seasonal activity and movements

of Necturus in Louisiana. Copeia 1967(4):732-737.

Wakeley, Jane F., G. J. Fuhrman, F. A. Fuhrman, H. G. Fischer and H. S.

Mosher. 1966. The occurrence of tetrodotoxin (tarichatoxin) in Amphibia

and the distribution of the toxin in the organs of newts (Taricha). Toxicon

3:195-203.

Webster, Dwight A. 1960. Toxicity of the spotted newt, Notophthalmus viri-

descens, to trout. Copeia 1960(l):74-75.

82 Ronald A. Brandon and James E. Huheey

Whitear, Mary. 1977. A functional comparison between the epidermis of fish

and of amphibians, Pp. 291-313 in R.I.C. Spearman (ed.). Comparative

Biology of Skin. Symp. Zool. Soc. Lond. 39. Academic Press, London. 427

pp.

Accepted 3 December 1981

Field and Laboratory Observations on

Microhabitat Selection, Movements, and Home

Range of Necturus lewisi (Brimley)

Ray E. Ashton, Jr. '

North Carolina State Museum of Natural History,

P. O. Box 27647, Raleigh, North Carolina 27611

ABSTRACT. — Movements, microhabitat selection and home ranges

of 20 adult and 5 juvenile Necturus lewisi were studied in the Little

River, Wake and Johnston counties, North Carolina, from November

1977 to May 1981. They were tagged with 6° Co wires and monitored

biweekly. Necturus lewisi occupied home ranges (x = 17.37m2 for

females and 73.25m2 for males). Five juveniles tagged and released

remained within 134 m2 over an eight-month period. The primary

microhabitat used by both juveniles and adults was loose granite

boulders on sand-gravel substrate. The next most important microhab-

itat was under bedrock embedded in stream banks. Leaf beds, reported

to be important habitat for N. lewisi, rarely were visited by the anim-

als. Microhabitat use varied with season and temperature range.

Trends indicated that dissolved oxygen, flow rate, and precipitation

influenced overall movement and microhabitat selection.

Retreat areas were maintained by juveniles and adults in the

laboratory, and similar behavior was observed in the field. The animals

moved sand and gravel by shoveling with the snout and transporting it

by mouth. Retreat areas were defended by adults, who displayed dis-

tinct threats and occasional attacks on intruders of either sex. Females

permitted males to cohabit their retreats during late winter and early

spring, when controlled laboratory temperatures were 8 to 14 degrees

C. Larvae and juvenile N. lewisi were not attacked and were permitted

to enter retreat areas unmolested. Both visual and olfactory cues were

used to locate and capture food. The primary method of feeding was

to sit at the mouth of the retreat where prey could be detected when it

came near. The animals commonly stalked prey at night. Courtship

was observed and was similar to that described for N. maculosus.

INTRODUCTION

Few studies have been published on Necturus behavior, move-

ments, and microhabitat use. Eycleshymer (1906), Smith (191 1), Bishop

(1926, 1941), and Harris (1961) reported on these topics as observed in

northern populations of Necturus maculosus. Cagle (1954), Shoop

(1965), and Shoop and Gunning (1967) made detailed studies of N.

1 Present address: International Expeditions, 1776 Independence Court, Birm-

ingham, Alabama 35216

Brimleyana No. 10:83-106. February 1985. 83

84 Ray E. Ashton, Jr.

maculosus and N. beyeri in Louisiana. Parzefall (1980) studied behavior

in N. maculosus and Proteus anguinus. Brimley (1924) briefly noted

that Necturus lewisi was found in leaf beds along fast moving waters.

Neill (1963) speculated that N. lewisi, like its close relative N. beyeri,

utilized bottom debris for cover. Ashton and Braswell (1979) provided

descriptions of a nest, larvae, and the distinctive post-hatchling larvae,

of N. lewisi.

The difficulty of capturing or locating animals frequently enough

throughout the year to determine general movements, microhabitat use

and home range appears to be the reason that such data are not availa-

ble for Necturus. Our three-year study attempted to remove this obsta-

cle by using 60Co tags, following methods described for use in sala-

manders by Barbour et al. (1969) and Ashton (1975), and in fish by Lee

and Ashton (1981). This study was conducted to obtain information on

behavior of N. lewisi in both its natural environment and the labora-

tory. Data on biotic and abiotic factors within the microhabitats used

by tagged animals were collected and analyzed in an effort to categorize

the species' general habitat requirements throughout the year. Observa-

tions on microhabitat use, feeding techniques, intra- and interspecific

interactions, and growth rates of hatchlings and adults, were made in

the laboratory.

METHODS AND MATERIALS

Field Studies

A preliminary study of N. lewisi in the Little River, a tributary of

the Neuse River in northern Wake County, began in November 1977.

The initial purpose was to develop methods of following movements,

determining home range, and studying other behavior using radioactive

tagging and tracking techniques. Three adults (two females, one male)

were caught in minnow traps, some the double-funneled wire type and

others employing plastic mesh. Each of the three animals was tagged

with two 60Co (35-50 mc) wires injected into the tail musculature. They

then were released at the site of capture, at least 20 m from each other.

We initially used a Thyac III survey meter and Model 491 scintilla-

tion probe to locate animals. However, with this equipment animals

were difficult to detect in more than 30 cm of water, making routine

monitoring of movements too sporadic. We also found that animals

may move more than 20 m, which necessitated a spatial separation of at

least 100 m if we were to recognize individuals without recapturing

them. Monitoring problems were overcome by acquiring more sensitive

Model 491 waterproof scintillation probes that permitted us to detect

animals at any depth at least 80 percent of the time.

From 12 November 1978 through 21 February 1979, 11 adult

animals were released at 150 to 250 m intervals along the stream (Site

N. lewisi Habitats and Behavior 85

No. 1). Two-hundred seventy-one positive sightings were made during

this period. Nine adult N. lewisi were captured and released within the

same 25 m2 area in Site No. 1 from 15 January 1980 to 28 August 1980.

We consequently were unable to identify individuals, but could monitor

microhabitat use and movement within the same area under similar

stream conditions. Two-hundred seventy-four sightings of tagged ani-

mals were made during this period. Similarly, on 21 February 1979 five

post-hatchling larvae, 41 to 46 mm total length (TL), were captured by

dip-netting, then were tagged and released in the Little River in Johns-

ton County (Site No. 2). One-hundred thirty-eight sightings were made

of these animals.

All tagged animals were monitored bi-weekly during all three study

periods. When a tagged animal was located, data were taken on ambient

temperature, flow rate, turbidity, dissolved oxygen, carbon dioxide, and

pH, both at the animal's current location and at the site where the

tagged animal had previously been found. Standard data were collected

at a single control station at each study site throughout the study. This

allowed us to evaluate changes in overall stream conditions and com-

pare them with varying conditions in the microhabitat. Weather data,

including maximum and minimum air and water temperatures and pre-

cipitation, were monitored at or near the control site. Other data were

obtained from the U.S. Weather Bureau, Raleigh-Durham Airport,

approximately 17 km from Site No. 1 and 27 km from Site No. 2.

The study areas were mapped for depth, bottom types, amount and

types of rock cover, and leaf bed development. Very little change in

these features took place in both areas throughout the study. General

water quality at both study sites was analyzed monthly from April to

December 1979 (Table 1). Water analysis was done using a Mach DR-

EL/2 with spectrophotometer.

Mercury reached peaks of 4.18 mg/ 1 (x=1.07) at Site No. 1 and

5.09 mg/ 1 (x=2.17) at Site No. 2. These levels indicated that mercury

was a potential pollutant. High levels of nitrates and sulfates were also

observed, notably in the May and June samplings, which followed peri-

ods of rain and subsequent runoff from surrounding farmlands. Con-

centrations of all other chemicals tested for were within normal limits.

Two surveys of aquatic invertebrates, one along a transect (Table 2)

and the other in a 10 m2 quadrat (Table 3), were made at Site No. 1 to

quantify potential food items in microhabitats used by N. lewisi. Aqua-

tic vertebrates (Table 4) were collected by seine, trap, and electroshock.

Standard SAS programs, including PROC-F reg, categorical data

and PROCORR using Perison, Sperman, and Kendall were used to

determine significance of specific correlations between behavior and

physical factors within the microhabitat. Although tests showed many

significant correlations between environmental factors, movement rate,

86

Ray E. Ashton, Jr.

Table 1

Water chemistry at Study Sites No. 1 (adult study area) and No.

(juvenile study area), April-December 1979.

Site No. 1

Site No. 2

and microhabitat selection, the ranges of comparable data were too

broad to make statistically valid statements on their significance.

Laboratory Studies

Larvae and post-hatchlings. — Eight larvae (41-47 mm TL) col-

lected on 21 February 1979 were maintained in 10-gallon aquaria con-

taining 6 cm of aerated water and 2 to 3 cm of sand-gravel substrate.

Small stones (x=4 cm2) were provided for cover. The larvae were fed

chopped red worms, chicken parts, and occasional aquatic invertebrates

and small ranid tadpoles. Growth was measured, and notes on color

changes and pattern were made, weekly. Observations were made on

feeding behavior, intra- and interspecific interactions, and shelter

manipulation.

Adults. — Fourteen adult N. lewisi were maintained in the laboratory

for from one to three years. They were kept in 50- and 85-gallon aquaria

with 4 to 6 cm of gravel substrate and containing granite rocks (6-15 cm

diameter) for cover. Four sets of 80x80x4 mm double plastic plates were

used as artificial cover. The top plate was sprayed with black latex paint

to block light, while the bottom plate was clear. The black plate could

be removed with little friction to permit observation of animals.

The study aquaria were maintained at 15° to 26° C (x=24°C) dur-

ing the first year. A temperature control unit used during the last two

N. lewisi Habitats and Behavior 87

years of the study permitted us to maintain variable water temperatures

equal to those observed at Site No. 1 (R=3°-24° C, x=16°C). Light sour-

ces were north-facing laboratory windows and overhead fluorescent fix-

tures. Light duration and intensity could not be controlled, but duration

was similar to normal seasonal day length. A red light was used for

some night observations.

DESCRIPTION OF STUDY SITES

Site No. 1

Site No. 1, where we studied adult N. lewisi, was located on Little

River at Mitchell's Mill Pond State Park, northern Wake County. This

was the same site used by Fedak (1971) during his study of the same

species. Little River is a headwater stream in the Neuse River drainage

of northeastern North Carolina. The stream begins on the Piedmont

Plateau and confluences with the Neuse River just after crossing the

Fall Line Zone.

Little River at Site No. 1 is a typical Piedmont stream; 30 percent

of the bottom and 8 percent of the banks are covered with granite

boulders and outcrops. The remainder of the stream bottom was sand

and fine gravel. The banks were steep, with a 3:1 or greater slope, and

consisted of sandy-clay soil. The banks were profusely pocked by bur-

rows of crayfish and other animals. On the high-energy side of the

stream the banks were undercut in a number of places, and burrows of

Castor and Ondatra were present in some. An average of 72 percent of

the main study area was shaded by the surrounding mixed deciduous

forest. Mean width of the stream at the control site for this area was 7

m, and mean depth was 1.2 m. The greatest depth recorded was 1.9 m,

although we estimated that water level may have reached a height of 2.4

m during severe flooding. The shallowest depth recorded at the control

site was 0.6 m. Water temperatures at the control site ranged between

8°C and 22° C during the spring and 1°C to 12°C during the winter.

The greatest change in water temperature between sightings (48 hr) was

5C°. Dissolved oxygen levels ranged between 4 ppm and 9 ppm (x=7.2

ppm). Turbidity ranged from 14 to 40 ftu (x=30.25 ftu). Mean non-

flood rate at the standard site was 4.9 cm/ second.

Site No. 2

Site No. 2, where we studied larvae, was in the Little River,

northwestern Johnston County, 12.8 km east of Site No. 1. This site lies

within the transitional Fall Line Zone between the Piedmont Plateau

and the Coastal Plain, as indicated by the lack of granite outcroppings,

presence of sandy soils, and paucity of large rocks in the river bottom.

Cypress trees, common along the river bank, were absent from the study

site itself. Twenty percent of the sandy bottom within Site No. 2 was

Ray E. Ashton, Jr.

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Table 4. Aquatic vertebrates collected or observed in Study Site No. 1

(1978-80).

Fishes

Acantharchus pomotis

Anguilla ro strata

Aphredoderus sayanus

Esox americanus

Erimyzon oblongus

Etheostoma olmstedi

Etheostoma vitreum

Gambusia affinis

Ictalurus natalis

Lepomis auritus

Lepomis cyanellus

Lepomis gulosus

Lepomis macrochirus

Micropterus salmoides

Nocomis sp.

Notropis altipinnis

Notropis amoenus

Notropis procne

Notropis sp.

Noturus insignis

Percina pelt at a

Percina roanoka

Umbra pygmaea

Amphibians

Reptiles

Amphiuma means

Desm ognathus fuscus

Eurycea bislineata

Necturus punctatus

Rana catesbeiana

Rana c lam i tans

Rana palustris

Acris gryllus

Bufo terrestris

Pseudemys concinna

Sternotherus odoratus

Nerodia sipedon

covered by small, flat, granite rocks with individual surface areas of not

more than 0.5 m2. Smaller flat, granite rocks (x=8 cm diameter) covered

approximately 15% of the bottom. Ten percent of the study area was

commonly covered with leaf beds during fall through early spring

months. The stream banks had a 2:1 slope, and were commonly under-

cut and pocked with numerous animal burrows.

Mean width of the stream at the standard control site for this area

was 13 m, with an average depth of 1.5 m. The greatest increase in

depth recorded was 2.4 m, although greater depths may have been

obtained during severe flooding when monitoring was not possible. The

shallowest depth recorded at the control site was 48 cm, with seasonal

fluctuations averaging +34 cm. Water temperatures at the standard site

ranged from 1°C to 21°C with seasonal fluctuations in temperature sim-

ilar to those at Site No. 1. Dissolved oxygen levels ranged from 5.4 ppm

to 8.0 ppm (x=7.2 ppm). Turbidity levels of 16 to 40 ftu (x=31.5 ftu)

were recorded. Table 1 summarizes the physicochemical data for both

sites.

N. lewisi Habitats and Behavior 93

RESULTS

Movements and Home Range of Adults

Of nine adult N. lewisi tagged in the first year, five were located

frequently enough (80% of all attempts) to permit measurement of home

range. However, even the tagged animals that were not located this fre-

quently appeared to move within a home range pattern. The size of the

ed by calculating the area within the outermost points of movement and

eterminwithin which 95 percent or more of all movements took place

(Table 5). Animals monitored in the second year, which were released

within a 25 m2 area, showed that home ranges overlapped regardless of

sex or season. Throughout the year all males made greater individual

movements (x=75.4 m) than females (x=17.5 m). Females displayed a

mean home range of 17.37 m2 while males had significantly larger home

ranges, x=73.25 m2 (Table 5).

Table 5. Home range and movements by adult Necturus lewisi.

Each home range contained bank areas with animal burrows or

rock overhangs, large flat rocks over a sand-gravel substrate, and slack

water areas where leaves and other debris formed detritus mats during

the fall and winter. Water depths ranged from 15 to 160 cm (x=73 cm)

(Table 6). We attempted to determine if movements of all animals

changed with environmental factors such as rainfall, barometric pres-

sure, moon phase, and air temperature. Seventy-two percent of all adult

movements took place within 24 hours after a rainfall of 9 mm or more.

Movements declined, however, when rainfall exceeded 40 mm. When

stream level increased by more than 15 cm, little movement was

observed unless the greater depth was maintained for more than 7 days.

In any season there was a 64 percent increase in overall movements as

barometric pressure fell or remained below 29.9 mm. Movements

increased when moon phase changed from full to dark. Overall move-

ments increased during the spring and fall and declined during the win-

ter and summer.

Movements correlated with overall stream and microhabitat tem-

perature changes. Carbon dioxide levels and pH remained relatively

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N. lewisi Habitats and Behavior 95

constant between the control site and any microhabitats used by N.

lewisi, and had no apparent bearing on movement.

Winter Movements and Microhabitat.— During winter, adult N

lewisi were found either under large granite rocks or in burrows in the

bank 85 percent of the time. Mean water temperature was 6.7° C. Of all

movements made during this period, 29 percent were to microhabitats

that were 1 C° or more higher than the standard site temperature (Table

6), and 28 percent were to microhabitats with higher levels of dissolved

oxygen (7-9 ppm) and higher flow rate (x= 14.79 cm/ sec). A decline in

overall number of movements was noted for all animals by 67 percent.

When the stream temperature dropped below 4°C, movement was to

areas where groundwater entered that was 2 to 4 C° higher than the

standard site temperature. No sexual difference in frequency of move-

ment was observed during the winter period.

Spring Movements and Microhabitat. — Between March and May

the number of movements was 48 percent greater than in all other sea-

sons. Generally, as temperature increased to 8°C, females showed

increased (27%) movement. At 14° C, female movement declined but

overall male movement increased. This period of increased male move-

ment coincided with expected breeding and nesting periods. Thirty-six

percent of movements made during this period were to microhabitats

with higher 02 levels (x=7.9 ppm) and 34 percent were to areas of

greater flow (x=7.8 cm/ sec). The primary microhabitats used during

period this were considered suitable nesting sites (Ashton and Braswell

1980). The microhabitats used 98 percent of the time by both sexes were

large bedrock outcrops or large boulders with sand and gravel beneath

them.

Summer Movements and Microhabitat . — Generally, summer move-

ments by both sexes, with an overall decline of 40 percent in frequency,

were to microhabitats that had lower temperatures (34%) and higher

oxygen levels (37%) (Table 6). The use of granite boulders and outcrops

in the main flow of the stream increased during this period.

Fall Movements and Microhabitat. — The overall river environ-

ment, including the microhabitats used by N. lewisi, was relatively uni-

form in temperature, oxygen levels, and other physical factors. General

movements were from the main flow to the winter retreat areas.

Movements and Microhabitats of Larvae and Post-hatchlings

Since five post-hatchlings were captured and released in the same

area, movements by specific individuals could not be determined. All,

however, remained within a 134 m2 area. At no time were two tagged

individuals found under the same cover, but it was not uncommon to

find three or more individuals within one square meter of one another.

96 Ray E. Ashton, Jr.

The primary habitat used by juveniles consisted of granite boulders, 0.5

m diameter, with underlying sand-gravel substrate. Movements into leaf

beds were rare (only two sightings) until early spring, when 17 percent

of all sightings were made in this habitat. The beds that salamanders

used were formed over a mud bank on the low-energy side of the river.

During this period, leaves were intact or only slightly decomposed, and

many potential food invertebrates were present in the leaf litter.

Captive Behavior

Retreats. — As did tagged animals in the field, captive N. lewisi

used rocks for cover or hid under artificial plexiglass plates when rocks

were not provided. Development of a retreat began within 24 hr after

the animal was placed in an aquarium. Retreats were made under cover

by moving the underlying sand and gravel to either side of the develop-

ing cavity, or by forcing it from under the rock. Substrate was removed

by shoveling or pushing with the snout and head. Excess sand and

gravel was moved through an opening that eventually became the main

opening to the cavity. This excess material formed an elliptical shelf

directly in front of the opening. The cavity itself usually was oval in

shape, and had a diameter measuring approximately two-thirds the total

length of the animal.

Once developed, the retreat area was maintained by the attending

animal. Gravel and sand were occasionally moved from the cavity by

mouth or by shoveling with the snout. Oral intake of gravel was

observed in three females and one male. The pieces, approximately 3

mm diameter, were moved to the shelf in front of the main entrance and

expelled from the mouth with some force. A female was observed

repeatedly picking up the same piece of gravel in the mouth, then plac-

ing it in a different location each time (like a bird placing sticks in a

nest). The shelf areas of all captives were maintained devoid of algae

and any other debris that commonly formed on surrounding gravel. The

rock surface directly over the cavity was similarity maintained.

In order to test cleaning behavior (?) by N. lewisi, flat granite rocks

with algae encrusted upper surfaces were collected from the Litte River.

They were then placed in a tank, on sand and gravel substrate, with

their encrusted surfaces facing down. Three adult N. lewisi were released

into the tank and established residence under the rocks. Within 48 hr,

the area of rock undersurface that covered each retreat was clear of

algae.

In microhabitats used by "tagged" animals in the field we on sev-

eral occasions observed similar retreat area maintenance. When animals

were under broken granite rocks the opening was always on the down-

stream side of the rock. The shelf area in front of the opening was

N. lewisi Habitats and Behavior 97

developed in a fashion similar to that previously described, and the

underside of the rock was devoid of encrustation.

Retreat development in larvae was not evident, although individu-

als repeatedly used the same rocks. Retreat development was apparent

in juveniles of more than 47 mm total length. The post-hatchling larvae

abandoned their retreats if larger rocks were placed in the aquaria.

Adults, however, remained in their original retreats even when addi-

tional cover was provided. Exceptions to this behavior were seen only in

those adults that were originally provided with plastic plates alone.

They would move to rock cover within 24 hr after it was introduced.

Feeding. — All captives were fed twice weekly and maintained on a

diet of earthworms, chopped chicken hearts, and live invertebrates,

along with occasional ranid tadpoles and minnows.

Both juveniles and adults displayed two feeding techniques. Most

commonly, an animal would lie in wait with snout protruding at the

opening of the retreat. Any organism or small object moving with the

flowing water that crossed the shelf area stimulated an alert response

from the attending animal, which flared its gills and moved partly out of

the opening. The second feeding technique was to actively search the

bottom of the tank. This was done primarily at night but sometimes

during the day. Distinct color fading was observed in striped post-

hatchling larvae when they were actively feeding at night. This fading

was so acute that their dark sides completely faded to gray-brown and

were indistinguishable from the light brown dorsum.

Both sight and olfaction apparently play an important role in locat-

ing food. Movement of prey could be discerned at least a meter away.

Movement of potential prey also stimulated, but was not necessary for,

an attack to be initiated. Feeding animals seemed to respond to very

slight movements when the prey was within 5 cm.

Olfactory response to food was tested by dropping chopped earth-

worms and chicken hearts at varying distances downflow and out of the

field of sight of the test animal. In 10 tests, where food was 1 m from it,

the animal responded within 32 to 125 seconds (x=47 sec). Response was

marked when the animal raised its head approximately 45 degrees from

the surface. As it walked towards the food, the animal would stop and

repeatedly put its snout to the substrate until it reached the food. Other

foods dropped on the shelf about 10 cm beyond the waiting animal

stimulated a similar response. Juveniles and larvae did not respond to

nonliving food dropped away from the entrance to their retreat areas.

Their response seemed to be stimulated by sight.

Active stalking of prey occurred at night. Prey included earth-

worms, Eurycea larvae, tadpoles, and the fishes Notropis, Etheostoma,

and Umbra. The mudpuppy would walk slowly in the direction of the

98 Ray E. Ashton, Jr.

prey, pausing frequently and putting its snout to the substrate. When

within striking distance, ca. 2 to 3 cm away, the animal would stop,

apparently watching the prey. At the slightest movement the mudpuppy

would engulf the prey with a rapid pharyngeal intake similar to that

described in Gyrinophilus (Bishop 1943; Cooper and Cooper 1968). A 4

cm earthworm or 3 cm fish would be totally and instantly engulfed, then

swallowed. Larger prey were often regurgitated and re-swallowed two or

more times before they were ingested whole. When larger prey was

taken, the mudpuppy would return to its retreat before regurgitating

and swallowing occurred. Fish were swallowed tail first. There was no

indication that the mudpuppies had difficulty with fin spines or the

sharp operculum of Etheostoma. The largest Notropis swallowed was

ca. 4 cm long. Fish were captured at night as they settled on the bottom.

The mudpuppy would stalk the intended prey, "freezing" each time the

fish moved, until it was within striking distance.

Courtship. — Courtship was observed only once, on 8 March 1979.

The female (146 mm SVL) had been in captivity three months. The male

(106 mm SVL) was released into the tank within two hours of capture.

Within twenty minutes after the male was introduced courtship was in pro-

gress. Initial contact between the two was not observed, but apparently

began at the female's retreat rock. The courtship took place within a 1

m2 area and continued for nearly an hour. The female was first observed

crawling slowly over the substrate with the male following ca. 2 to 4 cm

from her tail. The male would frequently touch its snout to the surface.

When the female stopped moving, the male moved forward and positi-

oned its snout just behind the rear leg of the female. During this initial

pursuit the female's gills were distinctly flared while the male's were held

close to the neck (Fig. 1).

The female remained motionless, and the male moved across her

body at the base of the tail. Once their bodies were parallel, the male

began to stroke or rub the female with his chin. Stroking began on the

top of the head, and the male moved his chin posteriorly along the

female's neck and mid-dorsum, then back to the head. This stroking

movement occurred twelve times in five minutes. Each time the male's

chin came into contact with her neck or head, the female would raise

her head from a position parallel to the substrate to an angle of 30

degrees or more; her body was held rigid. At this point the gills of both

animals were flared and rapidly pulsating (Fig. 2). The entire chin-

rubbing phase lasted a total of 18 minutes with actual rubbing occurring

sporadically at intervals of a minute or more. The stroke pattern

shortened in length to where the male's chin moved from first behind

the head onto the neck region. During this and subsequent phases, the

female remained motionless and kept her head slightly erect.

N. lewisi Habitats and Behavior

99

Fig. 1. Initial pursuit by male of female during courtship.

During the second phase the male slowly circled the female in a

clockwise movement. During circling, body contact was maintained

with the female (Fig. 3). As the male moved in front of the female her

head became more erect. Three complete circles were made by the male

in six minutes.

The courtship display ended when the male, on the third circling,

came into a parallel position with the female. The sides of their bodies

were touching, with the male's hind and foreleg resting on the female's

dorsum and their heads and tails parallel (Fig. 4). Both animals remained

in this position for more than 30 minutes, after which they retired to the

Fig. 2. Chin rubbing by male over

head and dorsum of female.

Fig. 3. Male circling female.

100

Ray E. Ashton, Jr.

Fig. 4. Male lying next to female; note position of legs.

female's retreat. The male followed directly behind the female. No

actual mating or spermatophore deposition was seen. Since the animals

could not be observed in the retreat the rock was carefully lifted, but

both animals became alarmed and left the area. The male remained with

the female for three days, after which it took up residence under another

rock. Following the male's departure the female was on one occasion

observed in an inverted position as though preparing to deposit eggs.

No eggs were deposited, however, and on the day following this final

observation the egg-laden female was found dead.

Aggression. — Females displayed greater territorial defense through-

out the year than did males, although males showed defensive postures

particularly in late spring and throughout summer. There were no

apparent differences in this behavior between the first year, when

temperatures were not regulated, and the two following years when

temperatures equal to those of the Little River site were maintained.

There were no apparent differences in the way males and females

defended their territories.

The area defended by all animals included the retreat and shelf

area, and approximately 10 cm in all directions from the edge of the

rock. There was no indication that scent played a role in identifying an

intruder; the resident responded only when visual contact was made.

N. lewisi Habitats and Behavior

101

There was some indication, however, that intruders may have used

olfaction in identifying an occupied territory. An intruder would touch

its snout to the gravel shelf or at the entrance. If the retreat was occu-

pied, the intruder usually moved hastily away from the area.

An animal in residence when an intruder approached made a threat

display. Upon observing the intruder, the resident moved out of the

retreat opening so that its head and gills were exposed. The gills would

flare to the maximum level and slowly pulsate (Fig. 5). If the intruder

moved away or stopped, the resident would retreat into the opening. If

the intruder instead moved toward the entrance or approached the shelf

area, the threat was repeated. The resident would move out of the

retreat if an intruder was on the shelf or close to the opening. At this

time the resident's gills would be flared and rapidly pulsating, and occa-

sionally its upper lip would be curled (Fig. 6). This was done quite

rapidly, and usually indicated that an attack was imminent. This lip curl

display also was quite commonly seen when adult animals were handled,

but no animals ever attempted to bite during capture or handling.

Attacks occurred in at least 50 percent of all intrusions observed.

Intrusions were quite rare, however, except when food or a new animal

was introduced into the tank. An attack would occur quite rapidly after

repeated threats and false charges. The resident animal would charge

Fig. 5. Initial threat display to intruder by resident animal.

102

Ray E. Ashton, Jr.

Fig. 6. Lip curl or flash. Upper lip is turned upward, revealing light underlying

tissue; gills flared.

forward and bite the intruder. Seventy percent of the bites were at the

base of the tail (Fig. 7), but some were just behind the foreleg. The bite

caused a circular wound of numerous lacerations, which usually left a

gray-white scar. Contact was fast and brief, and no attacking animal

was observed to hold onto its victim. A second or third bite was some-

times delivered as the intruder moved away. At no time was an intruder

seen to defend itself.

Occasionally an intruder would move unnoticed to a retreat open-

ing. If the intruder placed its head in or near the opening, the resident

animal would instantly bite the intruder's snout. Bites of this type were

similar to those previously described. The bite would include the area

Fig. 7. Attack, with biting at base of tail of intruder.

N. lewisi Habitats and Behavior 103

around the nostrils dorsally and the anterior end of the lower jaw ven-

trally. It was not uncommon for the intruder to thrash about while pull-

ing its head from the opening of the retreat.

Two larvae 49 mm and 51 mm TL, and two striped juveniles, were

introduced into a tank occupied by an adult so that adult-larval interac-

tions could be observed. Larvae were tolerated, and could venture into

retreats of either males or females. The adult animal became aware of

the larvae, possibly as potential food items, and often stalked them for

short distances. At no time, however, was a larva eaten, even when left

in the adult tank for two days. Gary Woodyard (pers. comm.) reported

a captive adult eating a juvenile, but the adult was not being regularly

fed.

Two adults, a male 84 mm SVL and a female 95 mm SVL, and two

subadults, 62 and 59 mm TL, of Necturus punctatus were released into

a tank with an adult N. lewisi to observe interspecific interactions. The

two subadult N punctatus were immediately eaten. The adults were

attacked by resident N. lewisi, even by individuals that were somewhat

smaller in body size than the N. punctatus. During these attacks the

threat displays were the same as previously described, but the biting was

distinctly different. Interspecific bites were directed to the head and gills

of the intruder. The attacker would grasp the victim until it thrashed

loose. On one occasion, an intruding N. punctatus was dragged to the

retreat opening and held there for several minutes. During this time

there was violent thrashing, including a rolling or spinning motion by

the N. lewisi. Wounds from these bites appeared to be much more

severe than those inflicted on intruding N. lewisi. Deep lacerations were

always evident, and covered the entire head and gill rakers if the head

had been engulfed, or were across the snout and throat region if the

head was not engulfed. These wounds often became infected and were

fatal.

Two N. lewisi, one juvenile 52 mm TL and one adult female 118

mm SVL, were introduced into a 10-gallon aquarium where two adult

N. punctatus were in residence. No attacks by the residents or by the

intruders were seen, although the N. lewisi went under rocks with resi-

dent N. punctatus. After one night the adult N. lewisi displaced one N.

punctatus. The displaced animal moved under, and remained under, the

rock with the other N. punctatus. The juvenile N. lewisi was moving

freely in the tank and never observed under rocks.

DISCUSSION

Necturus lewisi is a stream salamander that uses microhabitats

characterized by relatively high dissolved oxygen concentrations and

moderate stream flow. This is similar to other species of southern Nec-

turus that have been studied, including Necturus beyeri and Necturus

104 Ray E. Ashton, Jr.

maculosus, but considerably different from the wide spectrum of aqua-

tic habitats used by northern Necturus. The two primary microhabitats

used by N. lewisi in Piedmont Plateau environments are broken meta-

morphic or granite rock over sandy-gravel bottoms, and along stream

banks where there are rock outcroppings. This is contrary to the find-

ings of Fedak (1971), who stated that leaf beds are of primary impor-

tance. We found that leaf beds are only occasionally visited, probably

on feeding forays. Use of burrows or rock outcroppings is primary and

similar to those described by Neill (1963) for N. beyeri.

Rock microhabitats do not occur in the Coastal Plain range of N.

lewisi, and further studies of its ecology should be conducted in this

province. We can speculate, however, that bottom debris, roots, and

bank microhabitats replace rocks, and high oxygen levels and flow are

important factors in microhabitat selection. In any case, it would appear

that damming, stream clearing (snagging), or channelization would be

detrimental to N. lewisi.

Home ranges were observed, using tagged animals, and are similar

in size to those that Shoop and Gunning (1967) alluded to in their

mark-recapture studies of N. maculosus and N. beyeri. It appears that

male N. lewisi have a larger home range than females, and that they

have different and longer periods of movement. Changes in movement

and microhabitat use appears to be seasonally regulated by temperature

and rainfall. Environmental data were not collected in a way that would

provide statistically valid analyses.

Periodic high levels of mercury and other potential pollutants could

have long range effects on N. lewisi populations. However, there are no

data to show what these effects may be or at what concentrations detri-

mental effects take place (R. Hall, pers. comm.). The availability of such

data would be important in evaluating future conservation status of N.

lewisi.

Microhabitats are manipulated by N. lewisi. The animals actively

develop a retreat under cover and maintain the area free from algae and

debris. This was observed in the laboratory and in the field. The method

of cleaning sand and gravel by taking it into the mouth has not been

reported for this genus.

Pairing was observed in captive males during the breeding period,

and the more aggressive females permitted males to use the same retreat

areas at this time. Ashton and Braswell (1979) reported a male under a

nest rock and speculated that both males and females may participate in

nesting.

Agonistic behavior increased between individuals after the nesting

season. Threat displays in both sexes include flared and waving gills,

and lip curls. When two N. lewisi are involved, false attacks are often

displayed prior to actual contact. This is followed by a bite that appears

N. lewisi Habitats and Behavior 105

to cause little damage to the intruder. Attacks on N. punctatus, how-

ever, were more direct and resulted in serious wounds or death.

Parzefall (1980) reported that N. maculosus and Proteus anguinus

responded to olfactory cues in water and those left on substrates. Nectu-

rus maculosus recognized and avoided retreat areas occupied by Pro-

teus. Necturus lewisi displayed similar responses to retreats that were

not physically occupied by the resident. This indicates that similar olfac-

tory cues may be involved in identifying territory. Adult N. lewisi rec-

ognized larvae and juveniles and did not display territorial aggression

nor did they attempt to eat them.

Necturus lewisi was much more aggressive than the syntopic N.

punctatus, which never challenged intruding N. lewisi. Captive N. punc-

tatus were displaced by N. lewisi. This may indicate an amensalistic

relationship in the wild.

Courtship in N. lewisi was similar to that reported for N. maculo-

sus. Bishop (1941) observed that the courting male repeatedly crossed

over the female, which became motionless with head erect, but he did

not report chin rubbing or trailing as was observed in N. lewisi. These

may indicate that pheromones, as well as the tactile senses, play an

important sexual role. The actual transfer of spermatophores from male

to female is still unobserved in Necturus.

ACKNOWLEDGMENTS. — I express my appreciation to Keith

Everett and Ernie Flowers for their many long, cold, uncomfortable

hours in the field. I also thank Jesse Perry, Patricia Ashton, Dan Smith,

Paul Kumhyr, and the many others who assisted in the field. I thank,

too, Drs. John C. Clamp and John E. Cooper for identifying inverte-

brates, and further thank Dr. Cooper for the advice and assistance he

provided in all phases of the Necturus study. This study was partly

funded by a grant from the Carolina Conservationist Program, and con-

tract funds from a U.S. Fish and Wildlife Service (Office of Endangered

Species) cooperative agreement, both provided by the North Carolina

Wildlife Resources Commission. Other support was provided by the

North Carolina State Museum, a division of the N.C. Department of

Agriculture. Renaldo G. Kuhler, state museum scientific illustrator, did

the figures.

LITERATURE CITED

Ashton, Ray E., Jr. 1975. A study of movement, home ranges and winter behav-

ior of Desmognathus fuscus (Rafinesque). J. Herpetol. 9:85-91.

Ashton, Ray E., Jr., and A. L. Braswell. 1979. Nest and larvae of the Neuse

River Waterdog, Necturus lewisi (Brimley) (Amphibia: Proteidae). Brim-

leyana 1:15-22.

106 Ray E. Ashton, Jr.

Barbour, Roger W., J. W. Hardin, J. P. Shafer and M. J. Harvey. 1969. Home

range, movements and activity of the dusky salamander, Desmognathus

fuscus. Copeia 1969 (2):273-297.

Bishop, Sherman C. 1926. Notes on the habits and development of the mud-

puppy Necturus maculosus (Rafinesque). N.Y. State Mus. Bull. 268:5-60.

1941. The Salamanders of New York. N.Y. State Mus. Bull.

324. 268 pp.

Brimley, C. S. 1924. The Waterdogs {Necturus) of North Carolina. J. Elisha

Mitchell Sci. Soc. 40(3-4): 166-168.

Cagle, Fred R. 1954. Observations on the life history of the salamander Nectu-

rus louisianensis. Copeia 1954:257-260.

Cooper, John E., and M. R. Cooper. 1968. Cave-associated herpetozoa II:

Salamanders of the genus Gyrinophilus in Alabama caves. Natl. Speleol.

Soc. Bull. 30(2): 19-24.

Eycleshymer, Albert C. 1906. The habits of Necturus maculosus. Am. Nat.

40:123-36.

Fedak, Michael A. 1971. A comparative study of the life histories of Necturus

lewisi Brimley and Necturus punctatus Gibbes (Caudata & Proteidae) in

North Carolina. Masters thesis, Duke Univ., Durham. 103 pp.

Harris, John P. Jr. 1961. The natural history of Necturus, IV. Reproduction. J.

Grad. Res. Center 29:69-81.

Lee, David S., and R. E. Ashton, Jr. 1981. Use of 60 Co tags to determine

activity patterns of freshwater fishes. Copeia (3):709-71 1.

Neill, Wilfred T. 1963. Notes on the Alabama Waterdog, Necturus alabamensis

Viosca. Herpetologica 79(3): 166-174.

Parzefall, Jakob, P. Durand and B. Richard. 1980. Chemical communication in

Necturus maculosus and its cave-living relative Proteus anguinus (Protei-

dae, Urodela). Z. Tierpsychol. 53:133-138.

Shoop, Robert C. 1965. Aspects of reproduction in Louisiana Necturus popula-

tions. Am. Midi. Nat. 24:357-367.

, and Gerald C. Gunning. 1967. Seasonal activity and movements of

Necturus in Louisiana. Copeia 1967(4):732-737.

Smith, Bertram G. 1911. The nests and larvae of Necturus. Biol. Bull.

20(4): 191-200.

Accepted 20 August 1983

Pesticide and PCB Residues in the Neuse River Waterdog,

Necturus lewisi

Russell J. Hall

U. S. Fish and Wildlife Service,

Patuxent Wildlife Research Center, Laurel, Maryland 20708

Ray E. Ashton, Jr. '

North Carolina State Museum of Natural History,

P. O. Box 27647, Raleigh, North Carolina 27611

AND

Richard M. Prouty

U. S. Fish and Wildlife Service,

Patuxent Wildlife Research Center

ABSTRACT. — Residues of six organochlorine contaminants were

found in Necturus lewisi from six sites in the Tar and Neuse river

systems. Concentrations of pesticides were low and apparently related

to geographic patterns of use. Levels of PCBs were higher and did not

seem to vary geographically.

INTRODUCTION

The Neuse River Waterdog, Necturus lewisi, is a large, aquatic

salamander endemic to the Tar and Neuse river systems, North Caro-

lina. This paper reports the results of analysis of tissues to determine

pesticide and polychlorinated biphenyl (PCB) residue levels. This is only

the second published report of residues in salamanders, and the first

report that deals with an aquatic species. Some of the streams inhabited

by the salamander drain lands subject to frequent pesticide applications

(Reeves et al. 1977), and one of our sampling localities was the site of a

1979 PCB "spill".

Ten animals of various sizes were collected from two Coastal Plain

and three Piedmont Plateau localities in the Neuse River drainage.

Specimens were frozen soon after capture and shipped in dry ice from

Raleigh to the Patuxent Wildlife Research Center. Carcasses were pre-

pared for analysis by removal of gastrointestinal tracts. Either a 10-g

portion or the whole homogenized carcass was mixed thoroughly with

anyhdrous sodium sulfate, then extracted with hexane in a Soxhlet

apparatus for 7 hours* Extracts were cleaned up on a partially deacti-

vated Florisil column, and pesticides and PCB's were separated into

four fractions on a Silicar column (Kaiser et al. 1980).

1 Present address: International Expeditions, Inc., 1776 Independence Court,

Birmingham, AL 35216.

Brimleyana No. 10:107-109. February 1985. 107

108 Russell J. Hall, Ray E. Ashton, Jr., Richard M. Prouty

Residues were quantified using a gas-liquid chromatograph equipped

with an electron-capture detector and a 1.5% OV-17/ 1.95% QF-1 column

Residues in 10% of the samples were confirmed by mass spectrometry.

Recoveries of pesticides and PCBs from fortified tissues averaged 93%,

but residues reported were not corrected for recovery. The lower limit of

reportable residues was 0.01 ppm for pesticides and 0.05 ppm for PCBs.

All residue levels are expressed on a whole-body wet weight basis.

Pesticide residues in tissues (Table 1) were of low to moderate lev-

els and indicate contamination from a variety of sources. The presence

of DDT metabolites (DDD and DDE) and the absence of unaltered

DDT suggest that no recent sources of that discontinued pesticide exist

in the area. Residues of its metabolites probably are the result of appli-

cations made in past years. Both ds-chlordane and trans -nonachlor are

constituents of chlordane, a pesticide now used almost exclusively for

termite control.

Table 1. Residues of pesticides and PCBs in Necturus lewisi.

rnmnrt„n^i Geometric mean of Frequency of

Compound' residues (ppm)? occurrence

DDE

DDD

Dieldrin

cw-chlordane

fram-nonachlor

PCB 1254 __ ___

1 Heptachlor epoxide, oxychlordane, ds-nonachlor, endrin, toxaphene, HCB,

and mirex were not detected at the 0.01 ppm level of sensitivity.

2 Calculated on a whole-body wet weight basis; only those with measurable

residues were used in calculation of means.

Higher levels of DDE were found in Coastal Plain animals than in

those from Piedmont Plateau localities, and they occurred more fre-

quently in larger animals (> 30 g) than in smaller ones (< 30 g). Speci-

mens from the Coastal Plain averaged 0. 1 1 ppm DDE, while those from

the Piedmont averaged 0.02 ppm. Similarly, larger individuals averaged

0.15 ppm while smaller ones averaged 0.04 ppm. Two-way analysis of

variance shows that both trends are significant (p < 0.05). Dieldrin

residues were common in Coastal Plain specimens (frequency = 0.67),

but absent from Piedmont specimens. They also were more common in

larger specimens than in smaller ones (frequencies 0.6 and 0.2, respec-

tively). Components of chlordane showed a pattern similar to that of

dieldrin, with greatest contamination in specimens from lowland sites.

Pesticide and PCB Residues 109

Values for PCB residues, usually associated with industrial or municipal

wastes, were higher than those for pesticides. They tended to be consist-

ently high (range 0.2-1.2 ppm) in both Coastal Plain and Piedmont sites

and among animals of all sizes; no effect of the reported spill is evident.

A 1971 pesticide monitoring study (Reeves et al. 1977) was con-

ducted in Wayne County (Piedmont) and Wilson County (Coastal

Plain) localities that lie between our collecting sites. Their samples were

taken before the use of DDT was phased out, but neither DDT nor

dieldrin was applied in 1971 to the areas sampled. Therefore, levels of

DDT, its metabolites, and dieldrin should represent nearly maximum

background levels for tissues. Frogs sampled at that time had lower

residues than N. lewisi sampled in the same general area, eight years

later. Fish sampled in the 1971 study had higher residues, particularly of

the short-lived contaminants. In the only previous study of residues in

salamanders, Dimond et al. (1968) found in Maine that DDT and its

metabolites in Plethodon cinereus reached an average of 0.8 ppm soon

after the area was sprayed, and took eight or nine years to drop to

background levels (< 0.01 ppm).

ACKNOWLEDGMENTS.— We thank P. Freed, J. Reynolds, A.

Capparella, and E. Flowers, formerly of the N. C. State Museum

(NCSM), for assistance in various aspects of the project. J. E. Cooper,

E. H. Dustman, and J. C. Lewis edited drafts of the manuscript. The U.

S. Fish and Wildlife Service, Office of Endangered Species; the North

Carolina Wildlife Resources Commission; and the North Carolina State

Museum helped to fund work done by NCSM staff.

LITERATURE CITED

Dimond, J. B., R. E. Kadunce, A. S. Getchell and J. A. Blease. 1968. DDT

residue persistence in red-backed salamanders in a natural environment.

Bull. Environ. Contam. Toxicol. 5:194-202.

Kaiser, T. E., W. L. Reichel, L. N. Locke, E. Cromartie, A. J. Krynitsky, T. G.

Lamont, B. M. Mulhern, R. M. Prouty, C. J. Stafford and D. M. Swine-

ford. 1980. Organochlorine pesticide, PCB, and PPB residues and necropsy

data for bald eagles from 29 states— 1975-1977. Pestic. Monk. J. 75:145-149.

Reeves, R. G., D. W. Woodham, M. C. Ganyard and C. A. Bond. 1977. Preli-

minary monitoring of agricultural pesticides in a cooperative tobacco pest

management project in North Carolina, 1971— First year study. Pestic.

Monit. J. 77:99-106.

Ill

SUBSCRIPTIONS AND EXCHANGES

The editors anticipate two issues of approximately 150 pages each annually,

but may not always be able to adhere to this schedule. Subscription rates are for

the two forthcoming issues:

Individual— United States $ 9.00

Individual — Foreign $ 1 3.00

Institution $ 1 5.00

All subscriptions must be paid in advance.

Issues will be available on an exchange basis to organizations and institu-

tions publishing general natural history and ecology journals or papers on a

fairly regular schedule. Publications received on exchange will be placed in the

State Museum's H. H. Brimley Memorial Library.

Address all subscriptions and requests for information on purchase and

exchange to Managing Editor, Brimleyana, N. C. State Museum of Natural

History, P. O. Box 27647, Raleigh, NC 27611. Back issues are available.

DATE OF MAILING

Brimleyana No. 9 was mailed on 24 October 1984. (See below.)

PUBLICATION SCHEDULE

In periodicals that of necessity publish on an erratic schedule (such as Brim-

leyana), taxonomic and other priorities are established by the date of mailing of

an issue and not the putative date of publication on the cover or the dates of

acceptance of individual papers. International codes of nomenclature speak

clearly to this matter (see pp. 46-48 in DeBakey, Lois, et al. 1976. The Scientific

Journal. Editorial Policies and Practices. C. V. Mosby Co., St. Louis. 129 pp.).

Nevertheless, one of the major abstracting services and several of our subscrib-

ers have suggested that our publication schedule would be less confused (and

less confusing) if we eschewed the notion of "catching up" for the years 1983

and 1984. Since each issue of Brimleyana is numbered consecutively rather than

by volume, we are able to adopt this sensible methodology, beginning with this

issue (No. 10). Our subscribers will still receive the two forthcoming issues

covered by their subscriptions.

ERRATA

Brimleyana No. 4 (December 1980):

page 43: abstract, line 5, change Robeson to Richmond;

page 44: line 2, same change;

Brimleyana No. 6 (December 1981):

page 46: fourth line from bottom of page, change "jive other species" to

"four other species".

112

TABLE OF CONTENTS

1982

Number 7

Arndt, Rudolf G. (see Rohde, Fred C.) 69

Batch, Donald L. (see Branson, Branley A.) 137

Beaver, Thomas D., George A. Feldhamer and Joseph A. Chapman.

Dental and Cranial Anomalies in the River Otter (Carnivora: Mus-

telidae) 101

Branson, Branley A. and Donald L. Batch. Distributional Records for

Gastropods and Sphaeriid Clams of the Kentucky and Licking River

and Tygarts Creek Drainages, Kentucky 137

Casterlin, M. E. (see Reynolds, W. W.) 55

Chapman, Joseph A. (see Beaver, Thomas D.) 101

Dodd, C. Kenneth, Jr. Nesting of the Green Turtle, Chelonia mydas

(L.) in Florida: Historical Review and Present Trends 39

Dolin, Pamela S. and Donald C. Tarter. Life History and Ecology of

Chauliodes rastricornis Rambur and C. pectinicornis (Linnaeus)

(Megaloptera: Corydalidae) in Greenbottom Swamp, Cabell

County, West Virginia Ill

Feldhamer, George A. (see Beaver, Thomas D.) 101

Gibbons, J. Whitfield and Raymond D. Semlitsch. Terrestrial Drift

Fences with Pitfall Traps: An effective Technique for Quantitative

Sampling of Animal Populations 1

Hoffman, Richard L. Rediscovery and' Distribution of Bembidion

plagiatum Zimmermann (Coleoptera: Carabidae) 145

Hogarth, William T. (see Schwartz, Frank J.) 17

Jones, R. L. Distribution and Ecology of the Seepage Salamander

Desmognathus aeneus Brown and Bishop (Amphibia: Plethodonti-

dae) in Tennessee 95

Laerm, Joshua, Lloyd E. Logan, M. Elizabeth McGhee and Hans N.

Neuhauser. Annotated Checklist of the Mammals of Georgia 121

Lindquist, D. G. (see Reynolds, W. W.) 55

Logan, Lloyd E. (see Laerm, Joshua) 121

McGhee, M. Elizabeth (see Laerm, Joshua) 121

Miller, Grover C. (see Mobley, Ronald W.) 61

Mobley, Ronald W. and Grover C. Miller. Helminths of Some Sea-

birds from North Carolina 61

Neuhauser, Hans N. (see Laerm, Joshua) 121

Reynolds, W. W., M. E. Casterlin and D. G. Lindquist. Thermal

Preferenda and Diel Activity Patterns of Fishes from Lake

Waccamaw 55

Rohde, Fred C. and Rudolf G. Arndt. Life History of a Coastal Plain

Population of the Mottled Sculpin, Cottus bairdi (Osteichthyes:

Cottidae), in Delaware 69

113

Schwartz, Frank J., William T. Hogarth and Michael P. Weinstein.

Marine and Freshwater Fishes of the Cape Fear Estuary, North

Carolina, and Their Distribution in Relation to Environmental

Factors 17

Semlitsch, Raymond D. (see Gibbons, J. Whitfield) 1

Spurlock, Beverly D. (see Taylor, Ralph W.) 155

Tarter, Donald C. (see Dolin, Pamela S.) Ill

Taylor, Ralph W. and Beverly D. Spurlock. A Survey of the Fresh-

water Mussels (Mollusca: Unionidae) of Middle Island Creek, West

Virginia 155

Weinstein, Michael P. (see Schwartz, Frank J.) 17

Woodyard, Gary W. Incisor Malocclusion in a Specimen of Sylvilagus

floridanus (Mammalia: Lagomorpha) from North Carolina 151

Number 8

Bogan, Arthur E. (see Parmalee, Paul W.) 75

Bogan, Arthur E. (see Starnes, Lynn B.) 101

Bryan, Hal (see Caldwell, Ronald S.) 91

Caldwell, Ronald S. and Hal Bryan. Notes on Distribution and Hab-

itats of Sorex and Microsorex (Insectivora: Soricidae) in Ken-

tucky 91

Counts, Clement L., III. Occurrence and Distribution of Land Snails

of the Family Polygyridae (Mollusca: Gastropoda: Pulmonata) in

West Virginia 145

DeMont, David J. Use of Lepomis macrochirus Rafinesque Nests by

Spawning Notemigonus crysoleucas (Mitchill) (Pisces: Centrarchi-

dae and Cyprinidae) 61

Eagleson, K. W. (see Penrose, David L.) 27

Handley, Charles 0.(see Pagels, John F.) 51

Jones, Carol S. (see Pagels, John F.) 51

Klippel, Walter E. (see Parmalee, Paul W.) 75

Lenat, David R. (see Penrose, David L.) 27

Lewis, Julian J. Systematics of the Troglobitic Caecidotea (Crustacea:

Isopoda: Asellidae) of the Southern Interior Low Plateaus 65

McComb, William C. and Robert L. Rumsey. Response of Small

Mammals to Forest Clearings Created by Herbicides in the Central

Appalachians 121

Page, Lawrence M., Michael E. Retzer and Robert A. Stiles. Spawn-

ing Behavior in Seven Species of Darters (Pisces: Percidae) 135

Pagels, John F., Carol S. Jones and Charles O. Handley. Northern

Limits of the Southeastern Shrew, Sorex longirostris Bachman

(Insectivora: Soricidae) on the Atlantic Coast of the United

States 51

Parmalee, Paul W., Walter E. Klippel and Arthur E. Bogan. Aborigi-

nal and Modern Freshwater Mussel Assemblages (Pelecypoda:

Unionidae) from the Chickamauga Reservoir, Tennessee 75

114

Penrose, David L., David R. Lenat and K. W. Eagleson. Aquatic

Macroin vertebrates of the Upper French Broad River Basin 27

Retzer, Michael E. (see Page, Lawrence M.) 135

Rumsey, Robert L. (see McComb, William C.) 121

Schultz, George A. Terrestrial Isopods (Crustacea: Isopoda: Onisoi-

dea) from North Carolina 1

Starnes, Lynn B. and Arthur E. Bogan. Unionid Mollusca (Bivalvia)

from Little South Fork Cumberland River, with Ecological and

Nomenclatural Notes 101

Stiles, Robert A. (see Page, Lawrence M.) 135

115

INDEX TO SCIENTIFIC NAMES

Numbers 7: and 8: (1982)

Ablabesmyia parajanta 8:44

Abudefuduf saxatilis 7:31

Acantharcus pomotis 7:28,74

Acanthostracion polygonia 7:33

Acanthurus chirurgus 7:31

Acer rubrum 7:72

Achirus

fasciatus 7:22

lineatus 7:22

Acipenser oxyrhynchus 7:25

Acris gryllus 7:6

Acroneuria

abnormis 8:41

carolinensis 8:41

georgiana 8:41

Actinonaias

carinata 7:157: 8:104

ligamentina 8:80,81,83,84,104

gibba 8:108,110

pectorosa 8:104,108,110

Aeschna 8:42

Agapetus sp. 8:42

Agkistrodon piscivorus 7:8

Alasmidonta

marginata 7:157,158; 8:104,108,110

minor 8:104,106

truncata 8:104,106

viridis 8:104,106,108,1 10,1 16

Alectis ciliarus 7:29

Allocapnia 8:35

spp. 8:41

Allogona profunda 8:153,154,155

Allonarcys sp. 8:41

Alloperla sp. 8:41

Alosa

aestivalis 7:22,25

mediocris 7:22,25

pseudoharengus 7:22,25

sapidissima 7:22,25

Alouatta palliata 7:108

Aluterus schoepfi 7:32

Amblema

plicata 8:82,85

plicata 7:157

Ambystoma

opacum 7:6

talpoideum 7:6,1 1,12,14

tigrinum 7:6

Amia calva 7:25

Ammodytes americanus 7:31

Anchoa

cubana 7:25

hepsetus 7:25

mitchilli 7:22,26

nasuta 7:26

Ancylopsetta quadrocellata 7:32

Anguilla rostrata 7:22,25,73

Anisotremus surinamensis 7:30

Anodonta 8:87,88

corpulenta 8:78

grandis 8:78,87,88,104,108,1 10

grandis 7:157

imbecillis 8:80,87,104,108,1 10

spp. 8:80

suborbiculata 8:80,87

Anolis carolinensis 7:7

Antennarius

ocellatus 7:27

radiosus 7:27

Antocha sp. 8:43

Aphredoderus sayanus 7:26,74

Archosargus probatocephalus 7:30

Arctopsyche irrorata 8:42

Arius felis 7:26

Armadillidium

nasatum 8:2,4,10,19,20,21-22,24,25

vulgare 8:2,5,11,19,20,22,24,25

Armadilloniscus ellipticus 8:2,4,6,12,

16,22

Astroscopus

guttatus 7:31

y-graecum 7:31

Atherix lantha 8:43

Atrichopogon sp. 8:43

Baetis

amplus 8:40

nr. intercalaris 8:40

116

spp. 8:40

tricaudatus 8:40

Baetisca

berneri 8:41,46

Carolina 8:41

Bagre marinus 7:26

Bairdiella chrysoura 7:30

Balaena glacialis 7:129

Balaenoptera edeni 7:129

Balistes capriscus 7:23,33

Bathgobius soporator 7:31

Bembidion

honestum 7:147

inaequale 7:146

lacunarium 7:148

nigrum 7:147

plagiatum 7:145-150

scopulinum 7:145,146

texanum 7:148

Bidessus sp. 8:42

Bison bison 7:132

Blarina

brevicauda 7:8; 8:51,94,96,98,124,125,

126,127,128,131

churchi 7:122

telmalestes 8:51

carolinensis 8:51,94,98

carolinensis 7:122

Boyeria vinosa 8:42

Brachycentrus 8:46

sp. 8:42

Brachycercus nitidis 8:41,46

Brevoortia

smithi 7:25

tyrannus 7:22,25

Brillia sp. 8:44

Brotula

barbata 7:23

barbatus 7:27

Brundinia eumorpha 8:44

Bufo

quercicus 7:6

terrestris 7:6

Caecidotea 8:65-74

alabamensis 8:65

antricola 8:66

beattyi 8:65,66

bicrenata 8:65,66,71,72

bicrenata 8:65-74

whitei 8:65-74

jordani 8:65,66

meisterae 8:65,69,70

stygia 8:65,68,69

whitei 8:65,66

Caenis sp. 8:41

Calamus

leucosteus 7:22

sp. 7:22

Callitriche palustris 7:72

Calopteryx sp. 8:42

Cambarincola (?) 8:45

Cambarus spp. 8:46

Campeloma 8:114

crassula 7:142

crassulum 8:114

integrum 7:142

ponderosa 7:142

rubrum 8:114

Canis

familiaris 7:130

latrans 7:130

lupus 7:132

rufus 7:130,132

Cantherhines pullus 7:33

Caranx

bartholomaei 7:29

crysos 7:29

hippos 7:29

latus 7:29

Carcharinus

acronetus 7:24

falciformis 7:24

limbatus 7:24

obscurus 7:18,24

plumbeus 7:24

Cardiocladius sp. 8:44

Caretta caretta 7:41

Carunculina 8:106,107

lividus 8:106

Caryasp. 8:122

Castor canadensis carolinensis 7:126

Cemophora coccinea 7:8,1 1

Centrarchus macropterus 7:28

117

Centropomus undecimalis 7:28

Centropristis

ocyurus 7:28

philadelphica 7:28

striata 7:28

Centroptilum 8:40

nr. Centroptilum 8:40,46

Cephalanthus occidentalis 7: 1 12

Cernotina 8:43

Cervus

dama 7:131

elephus 7:132

Cetorhinus maximus 7:18,24

Chaetodipterus faber 7:30

Chasmodes bosquianus 7:31

Chauliodes

pectinicornis 7: 1 1 1 - 1 20

rastricornis 7:1 1 1-120

Chelonia mydas 7:39-54

Chelydra serpentina 7:7,12

Cheumatopsyche spp. 8:42

Chilomycterus schoepfi 7:33

Chironomus sp. 8:44

Chloroscombrus chrysurus 7:29

Choanotaenia sp. 7:64

Chologaster cornuta 7:26

Chrysops sp. 8:43

Cinygmula subaequalis 8:40

Citharichthys

macrops 7:32

spilopterus 7:32

Cladotanytarus sp. 8:44

Clethrionomys gapperi carolinensis

7:128

Cnemidophorus sexlineatus 7:7,13

Cnephia mutata 8:43

Coluber constrictor 7:8,12

Conchapelopia gr. 8:44

Condylura

cristata 7:8,13

parva 7:123

Conger oceanicus 7:22,25

Contracaecum sp. 7:63,64

Corbicula 8:111,114

fluminea 7:156; 8:110,113

Cordites sp. 8:44

Corydalus cornutus 8:43

Corynoneura spp. 8:44

Cottus 7:76,83,86; 8:142

bairdi 7:69-94

pygmaeus 7:76

Crangonyx sp. 8:46

Cricotopus (?) acutilabis 8:45

Cricotopus (C.) tremulus gr. 8:44

sp. 1 (=infuscatus?) 8:44

sp. 2 8:44

nr. flavocinctus 8:44

Cricotopus/ Orthocladius group 8:44,48

Crotalus horridus 7:8

Cryptochironomus fulvus gr. 8:44

Cryptotis

parva 7:8,123; 8:96,97

floridana 7:123

parva 7:123

Cultus decisus 8:41

Cura foremanii 8:46

Cyclonaias tuberculata 8:82,86,104,

108,110

Cyclisticus convexus 8:2,5,10,19,20,24

Cynoscion

nebulosus 7:30

nothus 7:30

regalis 7:22,30

Cyprinodon variegatus 7:27

Cyprinus carpio 7:26

Cyprogenia irrorata 8:80,83,87

Cypselurus exsiliens 7:27

Cystophora cristata 7:131,132

Dactylopterus volitans 7:32

Dasyatis

americana 7:24

centroura 7:24

sabina 7:22,25

sayi 7:25

violacea 7:22

Dasypus novemcinctus mexicanus 7:125

Deirochelys reticularia 7:7,12

Demicryptochironomus gr. 8:44

Desmognathus 7:95

aeneus 7:95-100

fuscus 7:97

monticola 7:97

ochrophaeus 7:97

wrighti 7:95

118

Diadophis punctatus 7:8

Diamesa spp. 8:44

Diapterus

auratus 7:22,30

olisthostomus 7:22

Dicranota sp. 8:43

Dicrotendipes sp. 8:44

Didelphis virginiana

pigra 7:122

virginiana 7:122

Dineutes sp. 8:42

Diplectrona modesta 8:42

Diplectrum formosum 7:28

Dixa sp. 8:43

Doliphilodes sp. 8:42

Dormitator maculatus 7:31

Dorosoma

cepedianum 7:25

petenense 7:25

Dromus

dromas 8:80,81,83,105

caperatus 8:105,109

Dugesia tigrina 8:46

Dysnomia 8:85,106,107

brevidens 8:104,107

capsaeformis 8:104

florentina walkeri 8:104

haysiana 8:105

triquetra 8:105

Eccoptera xanthenes 8:41

Echeneis naucrates 7:29

Ectopria nervosa 8:42

Elaphe obsoleta 7:8

Elaphropus vivax 7:146

Elassoma

evergladei 7:28

zonatum 7:28

Eliotris pisonis 7:31

Ellipsaria lineolata 8:80,88,105,109

Elliptio

crassidens 8:80,8 1 ,82, 104, 107, 108

dilatata 7:157,158

dilatatus 8:81,82,104,108,111,112

Elops saurus 7:25

Enneacanthus

chaetodon 7:28,55,56,57,58,59

gloriosus 7:28,58,59,74

obesus 7:28

Epeorus (Iron) 8:35,39

sp. 2 8:40

spp. 8:40

Ephemera

blanda 8:41

guttalata 8:41

Ephemerella 8:46

(Attenella) attenuata 8:40

(Danella)

lita 8:40

simplex 8:40

(Drunella)

conestee 8:40

cornutella 8:40

lata 8:40

longicornis 8:40

tuberculata 8:40

walkeri 8:40

wayah 8:40

(Ephemerella)

berneri 8:40

catawba 8:41

crenula(?)8:41

dorothea 8:41

hispida 8:41

invaria gr. 8:41

rossi 8:41

rotunda 8:41

(Eurylophella)

bicolor 8:41

funeralis 8:41

temporalis gr. 8:41

(Serratella)

Carolina 8:41

deficiens 8:41

serrata 8:41

serratoides 8:41

Epinephelus

morio 7:28

nigritus 7:28

striatus 7:28

Epioblasma 8:85,107

brevidens 8:107

triquetra 7:157

Epoicocladius cf. flavens 8:45

119

Eptesicus fuscus

fuscus 7:124

osceola 7:124

Eretmochelys imbricata 7:39

Erimyzon

oblongus 7:73

sucetta 7:26

Esox

americanus 7:26,73

niger 7:26,73

Etheostoma

(Belophlox) okaloosae 8:136

(Boleichthys)

exile 8:136

fonticola 8:136

fusiforme 7:74; 8:136

gracile 8:136

microperca 8:136

proeliare 8:136

(Boleosoma) 8:141,142

longimanum 8:135,136

nigrum 7:22; 8:136

olmstedi 7:22,29,74,89,91,92; 8:136

perlongum 7:55,56,57,58,59

(Catonotus) 8:141,142

barbouri 8:135,136,137,140,141,142

flabellare 8:136

kennicotti 8:136

neopterum 8:136

obeyense 8:135,136

olivaceum 8:136

smithi 8:136

squamiceps 8:136

striatulum 8:136

virgatum 8:136

(Doration) stigmaeum 8:136

(Etheostoma)

blennioides 8:136

tetrazonum 8:136

variatum 8:136

(Ioa) vitreum 8:136

(Litocara) nianguae 8:136

(Nothonotus) 8:141,142

acuticeps 8:142

aquali 8:135,136,137,141,142

camurum 8:136

maculatum 8:136,141,142

microlepidum 8:135,136,142

moorei 8:142

rubrum 8:142

rufilineatum 8:136

tippecanoe 8:136

(Nanostoma)

barrenense 8:136

coosae 8:135,136

duryi 8:135,136,137,138,139

rafinesquei 8:136

simoterum 8:136,138,139

zonale 8:136

(Oligocephalus) 8:140

asprigene 8:135,136,137,140

caeruleum 8:136,140

ditrema 8:136,140

grahami 8:136,140

lepidum 8:136,140

radiosum 8:136

spectabile 7:58; 8:136,140

(Ozarka)

boschungi 8:136

trisella 8:136

Etheostoma

radiosum 7:58

(Vaillanta) chlorosomum 8:135,136,

137,139,140

(Villora) edwini 8:136

Etropus

crossotus 7:22,32

microstomus 7:22,32

Eucinostomus

argenteus 7:22,30

gula 7:30

lefroyi 7:22

Eukiefferiella

bavarica gr. 8:45

claripennis gr. 8:45

clypeata gr. 8:45

devonica gr. 8:45

discoloripes gr. 8:45

Eumeces

fasciatus 7:7

inexpectatus 7:7

laticeps 7:7

Euorthocladius 8:45

Euorthocladius group 8:48

120

Eurycea

bislineata 7:6,97

quadridigitata 7:6

Falsifilicollis altmani 7:64,65

Farancia

abacura 7:8

erytrogramma 7:14

Felis

concolor 7:132

coryi 7:131

rufus 7:131

floridanus 7:131

rufus 7:131

Ferrissia

rivularis 7:143

sp. 8:46

Fistularia tabacaria 7:28

Fraxinus pennsylvanica 7:72

Fulmarus glacialis 7:62

Fundulus 7:58

diaphanus 7:27

heteroclitus 7:27

lineolatus 7:22,27

luciae 7:27

majalis 7:27

notti 7:22

spp. 7:22

waccamensis 7:55,56,57

Fusconaia

barnesiana 8:82,87,104,107,108

flava 7:157

subrotunda 8:82,86,87, 104, 106, 108

Gambusia affinis 7:27

Gasterosteus aculeatus 7:28

Gastrophryne carolinensis 7:7

Geodromicus brunneus 7:146

Geomys 7:126

colonus 7:126

cumberlandius 7:126

fontanelus 7:126

pinetis 7:126

fontanelus 7:126,132

pinetis 7:126

Gerres cinereus 7:22

Gerris sp. 8:43

Glaucomys

volans 7:126

querceti 7:126

saturatus 7:126

Globicephala macrorhynchus 7:129

Glossoma nigrior 8:42

Gobiesox strumosus 7:23,27

Gobionellus

boleosoma 7:22,31

hastatus 7:31

oceanicus 7:22

shufeldti7:31

stigmaticus 7:31

Gobiosoma

bosci 7:31

ginsburgi 7:31

Goera sp. 8:42

Goniobasis

costifera 7:142

ebenum 8:114

semicarinata 7:142; 8:114

sp. 8:46

Gonomyia gr. 8:43

Gymnura micrura 7:25

Gyraulus

parvus 7:143

sp. 8:46

Gyrinus sp. 8:42

Habrophlebia vibrans 8:40

Haplophthalmus danicus 8:2,4,7,13,18,

24

Hastaperla 8:41

Heleniella sp. 8:45

Helicopsyche borealis 8:42

Helisoma

anceps 7:143

trivolvis 7:143

Hemiramphus brasiliensis 7:27

Hemistena lata 8:104,107,108

Heterandria formosa 7:27

Heterodon

platyrhinos 7:8

simus 7:8

Heterotrissocladius cf. marcidus 8:45

Hexagenia munda (?) 8:40

Hexatoma sp. 8:43

121

Hippocampus erectus 7:28

Histrio histrio 7:27

Homaeotarsus bicolor 7:146

Hydatophylax argus 8:42

Hydrobaenus sp. 8:45

Hydroporus spp. 8:42

Hydropsyche

betteni 8:42

demora 8:42

mississippiensis 8:42

Hyla7:13

chrysocelis 7:6

cinerea 7:6

crucifer 7:6

femoralis 7:6

gratiosa 7:6

Hyloniscus riparius 8:2,4,7,13,17,24

Hypleurochilus geminatus 7:31

Hyporhamphus unifasciatus 7:27

Hypsoblennius

hentzi 7:31

ionthas 7:31

Ictalurus 7:59

catus 7:23,26

furcatus 7:26

natalis 7:26,73

nebulosus 7:26,73

punctatus 7:26

Ilex opaca 7:72

Isogenoides nr. hansoni 8:41

Isonychia spp. 8:40

Isoperla

holochlora 8:41

namata 8:41

orata 8:41

cf. slossonae 8:41

transmarina 8:41

Justicia americana 8:111

Kinosternon subrubrum 7:7,11,12,13

Kogia

breviceps 7:129

simus 7:129

Kyphosus

incisor 7:22,30

sectatrix 7:22,30

Lactophrys trigonus 7:33

Lagocephalus laevigatus 7:33

Lagoclon rhomboides 7:30

Lampetra aepyptera 7:73,89,91,92

Lampropeltis getulus 7:8

Lampsilis

alata 8:105

fasciola 7:157; 8:83,87,105,109,110,

112

glans 8:104

gracilis 8:105

ligamentina gibba 8:104

multiradiata 8:105

orbiculata 8:80

ovata 8:83,105,109,1 11,1 12

cardium 8:105,107,109,1 10

ventricosa 8:105,107

perdix 8:104

picta 8:105

punctata 8: 105

radiata luteola 7:157,158

recta 8:105

trabalis 8:105

vanuximensis 8:105

ventricosa 7:157,158; 8:105

Lanthus parvulus 8:42

Larimus fasciatus 7:23,30

Lasionycteris noctivagans 7:124

Lasiurus

borealis borealis 7:124

cinereus cinereus 7:124

intermedius floridanus 7:124

seminolus 7:124

Lasmigona

complanata 7:157

costata 7:157; 8:82,105,108,110

holstonia 8:116

Lastena lata 8:104

Leiostomus xanthurus 7:22,30

Lemoix rimosus 8:83,87

Lepidostoma sp. 8:42

Lepisosteus 7:25

Lepomis

auritus 7:29

cyanellus 8:61

gibbosus 7:29,74

gulosus 7:29

macrochirus 7:29,58,74; 8:61-63

122

marginatus 7:55,56,57,58,59

microlophus 7:29

sp. 8:62

Leptodea

fragilis 8:88,105,109,1 10

laevissima 8:80

sp. 8:80

Leuctra sp. 8:41

Lexingtonea dolabelloides 8:82,84

Ligia

exotica 8:1,2,4,5,6,12,22

sp. 8:1,5

Ligidium

blueridgensis 8:1,2,4,8,14-16,23

elrodii 8:2,4,8,14,15,24,25

Limnodrilus hoffmeisteri 8:45

Limnophyes sp. 8:45

Limonia sp. 8:43

Lioplax

subcarinata occidentalis 7:142

sulculosa 7:142

Liquidamber styraciflua 7:72

Lirceus sp. 8:46

Liriodendron tulipifera 7:72; 8:122

Lissobiops serpentinum 7:146

Lithasia plicata 7:141

Lithasis obovata 7:141

Lophius americanus 7:27

Lucania

goodei 7:27

parva 7:27

Ludwigia palustris 7:72

Lutjanus

analis 7:29

griseus 7:30

jocu 7:30

synagris 7:30

Lutra

canadensis 7:101-109

lataxina 7:131

Lymnaea

columella 7:143

humilis 7:143

obrussa form 7:143

palustris 7:143

stagnalis 7:143

Lype diversa 8:42

Macronychus glabratus 8:42

Magnolia

acuminata 8:122

virginiana 7:72

Malirekus hastatus 8:41

Manta birostris 7:25

Marmota 7:151

monax monax 7:125

Matrioptila jianae 8:42

Medionidus conradicus 8:105,109,

110,111,112,116

Megalonaias gigantea 8:88

Megalops atlanticus 7:25

Megaptera novaeangliae 7:129

Membras martinica 7:27

Menidia 7:58

beryllina 7:27

extensa 7:55,56,57

menidia 7:27

spp. 7:22

Menticirrhus

americanus 7:30

littoralis 7:30

saxatilis 7:30

Mephitis

mephitis 7:131

elongata 7:131

nigra 7:131

Mesodon 8:149,151

albolabris

albolabris 8:155

dentatus 8:155

appressus 8:149,150,155

perigraptus 8:155

burringtoni 8:156

clausus 8:151,152,155

dentiferus 8:149,152,155

inflectus 8:151,152

mitchellianus 8: 1 5 1 , 1 52, 1 55, 1 56

panselenus 8:156

pennsylvanicus 8:151,152,155

perigraptus 8:149,152,156

profundus 8:155

rugeli 8:151,152

sayanus 8:149,150,155

thyroidus 8:148,149,155

bucculenta 8:156

123

thyroidus 8:156

zaletus 8:149,150,155

Mesoplodon

densirostris 7:129

europaeus 7:129

Micrasema wataga 8:42,46

Microdesmus longipinnis 7:23,31

Microgobius thalassinus 7:31

Micromya

nebulosa 8:105

picta 8:105

trabalis 8:105

vanuxemensis 8:105

Micropogonias undulatus 7:22,30

Micropsectra 8:47

spp. 8:44

Micropterus

dolomieui 8:61

salmoidess 7:29; 8:61

Microsorex 8:91-100

hoyi 7:132; 8:51,91,92,94-95,96,97

winnemana 7:122

thompsoni 7:122

Microtendipes sp. 8:44

Microtus

pennsylvanicus pennsylvanicus 7:128

pinetorum 7:9,128; 8:124,125,126,

128,130,132

auricularis 7:128

parvulus 7:128

pinetorum 7:128

Microvelia sp. 8:43

Miktoniscus

halophilus 8: 1 ,2,4,7, 13,17, 1 8,22,23,24

medcofi 8:1,2,4,8,13,17-18,24

Miliobatis freminvillei 7:25

Monocanthus 7:21

Morone

americana 7:28

saxatilis 7:28

Moxostoma

anisurum 7:22,26

macrolepidotum 7:26

pappillosum 7:22

Mugil

cephalus 7:23,31

curema 7:31

spp. 7:22

Mus musculus 7:128

Musculium 7:140

Mustela

frenata 7:130

noveboracensis 7:130

olivacea 7:130

vison 7:130

lutensis 7:131

mink 7:131

Mustelus canis 7:23,24

Mycteroperca

bonaci 7:28

microlepis 7:28

Myrophis punctatus 7:25

Myocastor coypus bonairiensis 7:129

Myotis

grisescens 7:123

keenii 7:123

septentrionalis 7:123

leibii 7:124

lucifugus lucifugus 7:123

sodalis 7:124

Nais

behningi 8:46

elinguis 8:46

simplex 8:46

Nanocladius

nr. balticus 8:45

spp. 8:45

Narcine brasiliensis 7:24

Napaeozapus insignis roanensis 7:128

Negaprion brevirostris 7:24

Neoephemera purpurea 8:41

Neofiber alleni exoristus 7:128

Neophylax spp. 8:42

Neotoma

floridana 7:9,127

floridana 7:127

haemotoreia 7:127

illinoensis 7:128

Nerodia

erythrogaster 7:7

fasciata 7:7

Neureclipsis sp. 8:43

Nigronia

fasciatus 8:43

124

serricornis 8:43

spp. 8:43

Nitocris trilineata 7:141

Notemigonus crysoleucas 7:26,73; 8:61-63

Notonecta undulata 7:1 15

Notophthalmus viridescens 7:6,13

Notropis

chalybaeus 7:26

cornutus 8:61

cummingsae 7:18,26

petersoni 7:26

umbratilis 8:61

Noturus 7:58,59; 8:142

gyrinus 7:26,55,56,57

sp. 7:55,56,57

Nuphar luteum 8:62

Nycticeius humeralis humeralis 7:124

Nyctiophylax nephophilus 8:43

Nyssa sylvatica 7:72

Obliquaria reflexa 8:88,89,105

Obovaria

circula 8:105

olivaria 8:80

retusa 8:83,87

subrotunda 7:157; 8:83,86,105,109,

110,112

Oceanites oceanicus 7:62

Ochrotomys

nuttalli 7:127; 8:124,125,126,128,130,

131,132

aureolus 7:127

nuttalli 7:127

Ochrotrichia sp. 8:42

Odocoileus

virginianus 7:132

nigribarbis 7:132

Odontomesa cf. fulva 8:44

Ogcocephalus rostellum 7:27

Oligoplites saurus 7:29

Ondatra zibethicus zibethicus 7:128

Oniscus asellus 8:2,4,8,13,19,24

Oostethus

brachyurus 7:28

lineatus 7:21

Ophichthus 7:21

cruentifer 7:23,25

gomesi 7:25

ocellatus 7:25

Ophidion

grayi 7:27

marginatus 7:21

welshi 7:21,22,23,27

Ophisaurus

attenuatus 7:7

ventralis 7:7

Opisthovarium elongatum 7:64

Opsanus tau 7:26

Optioservus sp. 8:42

Orthocladius 8:45

(Euorthocladius)

sp. 1-6 8:45

(Orthocladius)

nr. clarkei 8:45

nr. dorenus 8:45

cf. nigritus 8:45

cf. obumbratus 8:45

cf. robacki 8:45

Orthopristis chrysoptera 7:30

Oryzomys palustris palustris 7:126

Oulimnius latiusculus 8:42

Oxydendrum arboreum 8:122

Pagastia

orthogonia 8:47

sp. 8:44,47

Palpomyia (complex) 8:43

Parachaetocladius sp. 8:45

Parachironomus sp. 8:44

Paragnetina immarginata 8:41

Parakiefferiella sp. 8:45

Paralichthys

albigutta 7:32

dentatus 7:32

lethostigma 7:32

spp. 7:22

squamilentus 7:32

Paraphaenocladius sp. 1 & 2 8:45

Parapsyche cardis 8:42

Paratendipes sp. 8:44

Pegiasfabula 8:103,104,108,1 11,1 12

Peloscolex variegatus 8:45

Peltoperla 8:35

sp. 8:41

125

Perca flavescens 7:29,74

Percina

(Alvordius)

maculata 8:136

notogramma 8:136

peltata 8:136

(Cottogaster) copelandi 8:136

(Ericosoma) evides 8: 1 35, 1 36, 1 37, 1 38

(Hypohomus) aurantiaca 8:136

(Percina) caprodes 8:136

Peprilus

alepidotus 7:32

burti 7:32

triacanthus 7:32

Pericoma sp. 8:43

Perlesta sp. 8:41

Peromyscus 7:132

gossypinus 7:9,127

anastasae 7:127

gossypinus 7:127

megacephalus 7:127

leucopus 8:124,125,126,128,130,

131,132

leucopus 7:127

maniculatus nubitterae 7:127

polionotus 7:9

colemani 7:127

polionotus 7:127

subgriseus 7:127

Petromyzon marinus 7:24

Phaethon aethereus 7:62

Phasgonophora capitata 8:41

Philonthus sp. 7:146

Philoscia vittata 8:1,2,4,6,12,16,22

Phoca vitulina 7:132

Physa

gyrina 7:143

heterostropha 7:143

integra 7:143

Physellasp. 8:114

Pimephales 8:142

Pinus

echinata 8:122

rigida 8:122

Pipistrellus

subflavus 7:124

floridanus 7:124

subflavus 7:124

Pisidium

compressum 7:140

sp. 8:46

Pisodonophis 7:21

Pituophis melanoleucus 7:8

Plagiola 8:86,87,107

arcaeformis 8:83,85,86,104,108

capsaeformis 8:104,109

cf. capsaeformis 8:83,85,86

donaciformis 8:105

elegans 8:105

florentina 8:83,85,86

walkeri 8:104,109

flexuosa 8:83,85,86

haysiana 8:83,85,105,109

interrupta 8:83,85,86, 104, 107, 108

lewisi 8:86

lineolata 8:105

obliquata 8:83,85,86

propinqua 8:83,85,86

spp. 8:86

stewardsoni 8:83,85

torulosa 8:83,85,86

gubernaculum 8:86

torulosa 8:86

torulosa/ propinqua 8:83

triquetra8;83,86,105

turgidula 8:83,85,86

Plecotus

rafinesquii 7:124

macrotis 7:125

rafinesquii 7:125

Plectronemia 8:43

Plethobasus 8:85

cicatricosus 8:82,85

cooperianus 8:80,82,85

cyphus 8:80,82,85

Plethodon

glutinosus 7:6,97

jordani 7:10

Pleurobema 8:84

clava 7:157; 8:82,84,104,106

coccineum 8:104,108

cordatum 8:78,80,82,84,104,108

coccineum 8:104

cordatum 8:104

pyramidatum 8:104

crudum 8:104

126

oviforme 8:84,104,106,116

oviforme "complex" 8:104,106,

108,110

plenum 8:82,84

pyramidatum 8:82,84,104,108

spp. 8:81,82

Pleurocera

acuta 7:141; 8:1 14

canaliculatum 7:141

undulatum 7:141

Podostemon 8:35

Poecilia latipinna 7:27

Pogonias cromis 7:30

Polycentropus spp. 8:43

Polygonum

hydropiperoides 8:61

punctatum 8:61

Polymeda/Ormosia 8:43

Polypedilum

angulum 8:44

aviceps 8:44

convictum 8:44

fallax 8:44

halterale gr. 8:44

illinoense 8:44

laetum (?) 8:44

scalaenum 8:44

Pomatiopsis lapidaria 7:142

Pomatomus saltatrix 7:29

Pomoxis nigromaculatus 7:29

Porcellio

laevis 8:2,5,9,15,20-21,23,24

scaber 8:2,5,9,15,18,19-20,21,23,24

virgatus 8:2,5,6,20,21,23

Porcellionides pruinosus 8:2,5,8,12,21,24

Porichthys plectrodon 7:26

Potamilus 8:107

alata 8:105,109,1 11,1 12

alatus 8:83,88

spp. 8:80

Potamogeton sp. 7:72

Potthastia

cf. gaedi 8:44

nr. longimanus 8:44

Priacanthus

arenatus 7:29

cruentatus 7:29

Prionotus

carolinus 7:32

evolans 7:32

salmonicolor 7:32

scitulus 7:32

tribulus 7:32

Pristigenys alta 7:29

Pristina

idrensis 8:46

longiseta (?) 8:46

Procyon

lotor 7:130

elucus 7:130

litoreus 7:130

solutus 7:130

varius 7:130

Prodiamesa olivacea 8:44

Promoresia

elegans 8:42,46

tardella 8:42,46

Proptera 8:107

alata 8:105

Prosimulium mixtum 8:43,46

Prostoma graecens 8:46

Protoplasa fitchii 8:43

Psephenus herricki 8:42

Pseudacris

nigrita 7:6

ornata 7:7

Pseudemys

floridana 7:12

scripta 7:7,12

Pseudocloeon spp. 8:40

Pseudodiamesa pertinax 8:47

Pseudorca crassidens 7:129

Pseudostenophylax uniformis 8:42

Pseudotriton

montanus 7:6

ruber 7:6

Psychoda sp. 8:43

Pterodroma

hasitata 7:63

spp. 7:62

Ptychobranchus

fasciolare 8:105,109,1 13

phaseolus 8:105

subtentum 8:105,109,111,113

127

subtentus 8:105

Puffinus spp. 7:62

Pychopsyche

guttifer 8:42

lepida 8:42

Quadrula 8:84

coccinea 8:104

cylindrica 8:82,85,104,108

intermedia 8:82,85

metanevra 8:82,84

obliqua 8:104

pustulosa 8:80,82,85,104,108

pustulosa 7:157

pyramidata 8:104

quadrula 7:157

spp. 8:82

subrotunda 8:104

tritigonia 8:104

tuberculata 8: 104

Quercus

alba 8:122

coccinea 8:122

prinus 8:122

rubra 8:122

velutina 8:122

Rachycentron canadum 7:22,29

Raja eglanteria 7:24

Rana

areolata 7:7

catesbeiana 7:7

clamitans 7:7

utricularia 7:7,13

Rattus

norvegicus 7:128

rattus 7:128

Reithrodontomys

humulis 7:9

humulis 7:126

Remora remora 7:29

Rheocricotopus

cf. robacki 8:45

sp. 2 & 3 8:45

Rheotanytarsus 8:47

spp. 8:44

Rhimesomus bicaudalis 7:33

Rhincodon typhus 7:18,24

Rhinobatos lentiginosus 7:24

Rhinoptera bonasus 7:22,25

Rhizoprionodon terraenovae 7:24

Rhododendron maxium 7:97

Rhyacophila

acutiloba 8:43

amicis 8:43

atrata 8:43

Carolina 8:43

fuscula 8:43

nigrita 8:43

torva 8:43

Rissola marginata 7:21,22

Rithrogenia 8:35

sp. 8:40

Saetheria tylus 8:44

Salix nigra 7:112

Sardinella aurita 7:25

Scalopus

aquaticus 7:8,123

aquaticus 7:123

australis 7:123

howelli 7:123

Scaphiopus holbrooki 7:6,13

Sceloporus undulatus 7:7

Sciaenops ocellatus 7:23,30

Scincella laterale 7:7

Sciurus

carolinensis 7:151

carolinensis 7:125

niger 7:125

bachmani 7:125

rufiventer 7:126

shermani 7:126

Scomberomorus

cavalla 7:23,32

maculatus 7:32

Scopaeus sp. 7:146

Scophthalmus aquosus 7:32

Scorpaena brasiliensis 7:32

Selene

setapinnis 7:29

vomer 7:29

Seriola

dumerili 7:29

rivoliana 7:29

zonata 7:29

128

Setodes sp. 8:42

Sialis 7:115

sp. 8:43

Sigmodon

hispidus 7:9,127

hispidus 7:127

komareki 7:127

Simpsonaias ambigua 7:157

Simulium

(Phosterodoros)

decorum 8:43

spp. 8:43,46

vittatum gr. 8:43

Skrjabingylus sp. 7:102

Sorex 8:91-100

cinereus 7:132; 8:55,56,91,92,93,

96,97,98

cinereus 7:122; 8:92,93

fontinalis 8:55

lesueurii 8:92,93

dispar 7:132; 8:51,91,92,94,96,98

fumeus 8:91,92,94,96,98,124,125,

126,127,128,131

fumeus 7:122

hoyi8:51

longirostris 7:122; 8:51-59,91,92,

93,96,97,98

fisheri 8:52,55

longirostris 7:122; 8:51-59

palustris 8:51,91,95,96

Sparganium sp. 7:72

Specaria josinae 8:46

Sphaerium 7:140; 8:46

corneum 7:140

fabale 7:140

lacustre 7:140

nitidum 7:140

simile 7:140

striatinum 7:140

transversum 7:140

Sphoeroides

maculatus 7:33

spengleri 7:33

Sphyraena

barracuda 7:31

borealis 7:31

guachancho 7:31

Sphyrna

lewini 7:22,24

tipuro 7:24

Spilogale putorius putorius 7:131

Squalus acanthias 7:22,23,24

Stellifer lanceolatus 7:23,30

Stenacron

interpunctatum 8:40

pallidum/ Carolina 8:40

Stenella plagiodon 7:129

Stenelmis sp. 8:42

Steno bredanensis 7:129

Stenonema

carlsoni 8:40

ithaca 8:40,46

modestum 8:40

pudicum 8:40

terminatum (?) 8:40

Stenotomus chrysops 7:30

Stenotrema 8:153

barbigerum 8:153,154

burringtoni 8:156

edvardsi 8:153,154,155

fraternum 8:153,154,155

cavum 8:155

fraternum 8:155

hirsutum 8:151,152,154,155,156

leai 8:153,154

simile 8:155

stenotrema 8:153,154,155

Stephanolepis hispidus 7:23,33

Sterna

anaethetus 7:62,63

hirundo 7:62

Sternotherus odoratus 7:7,11,12

Stictochironomus sp. 8:44

Storeria

dekayi 7:7

occipitomaculata 7:8

Strongylura marina 7:27

Strophitus

edentulus schaefferinana 8:104

undulatus 8:104,109,110

undulatus 7:157

cf. Strophitus undulatus 8:82

Strophopteryx sp. 8:41

Stylurus scudderi 8:42

129

Susscrofa 7:99,131

Sweltsa sp. 8:41

Sylvilagus

aquaticus aquaticus 7:125

floridanus 7:9,151-153

mallurus 7:125

palustris palustris 7:125

transitionalis 7:125,132

Symphitopsyche

bronta 8:42

macleodi 8:42

morosa 8:42

sparna 8:42

Symphurus

civittus 7:23,32

plagiusa 7:32

Symphynota costatus 8:104

Synaptomys cooperi 7:132

Syndiamesa zavreli 8:44

Syngnathus

floridae 7:28

fuscus 7:28

louisianae 7:28

springeri 7:28

Synodus foetens 7:26

Synorthocladius sp. 8:45

Tabanus sp. 8:43

Tadarida brasiliensis cynocephala 7:125

Taeniopteryx 8:35

burksi 8:41

Tamias

striatus 8:124,125,126,127-130,131,132

striatus 7:125

Tamiasciurus hudsonicus abeiticola 7:126

Tantilla coronata 7:8,11,13

Tanytarsus

spp. 8:44

(Sublettea) coffmani 8:44

Tautoga onitis 7:31

Terrapene Carolina 7:5,7

Tetrabothrius

filiformis 7:64,65

laccocephalus 7:64,65

minor 7:64,65

procerus 7:64,65

sp. 7:64

Thamnophis

sauritus 7:8

sirtalis 7:8

Thienemaniella spp. 8:45

Tipula spp. 8:43

Toxolasma 8:106,107

lividus 8:104,106,107,108,110

Trachinotus

carolinus 7:29

falcatus 7:29

Trachurus lathami 7:29

Trepobates sp. 8:43

Tribelos jucundus 8:44

Tribonema 7:114

Trichechus manatus latirostris 7:131

Trichiurus lepturus 7:31

Trichoniscus pusillus 8:2,4,8,13,17,24

Trinectes maculatus 7:22,32

Triodopsis 8:147

albolabris 8:147,148,155

denotata 8:146,147,155

dentifera 8:155

fallax 8:155

fraudulenta 8:155

fraudulenta 8:147,148,155

vulgata 8:147,148,155

juxtidens

juxtidens 8:155

discoidea 8:155

multilineata 8:156

platysayoides 8:155

rugeli 8:155

rugosa 8:147,148,155

tridentata 8:146,147,155

juxtidens 8:155

rugosa 8:155

tridentata 8:155

Tritigonia verrucosa 8:104,108

Tritogonia verrucosa 7:157,158; 8:88

Truncilla 8:106,107

arcaeformis 8:104

brevidens 8:104

capsaeformis 8:104

donaciformis 8:105,109

haysiana 8:105

triquetra 8:105

truncata 8:105,109

130

walked 8:104

Tursiops truncatus 7:129

Umbra pygmaea 7:26,73

Unio

crassidens 8:104

gibbosus 8:104

Urocyon

cinereoargenteus 7:130

floridanus 7:130

Urophycis

earli 7:27

floridana 7:27

regia 7:27

Ursus

americanus 7:130

floridanus 7:130

Vaccinium 8:57

Villosa

fabalis 7:157

iris 8:105,109,1 10,1 13,1 16

iris 7:157

taeniata 8:113,116

picta 8:105,109

punctata 8:105,109,110

trabalis 8:105,109,110,116

vanuxemensis 8:105,106,109

cf. venuxemi 8:83

venuxemi/P. capsaeformis/

P. florentina

Virginia

striatula 7:8

valeriae 7:8

Vulpes vulpes fulva 7:130

Wormaldia sp. 8:42

Zalophus californianus 7:131

Zapus hudsonius americanus 7:128

Zavrelia sp. 8:44

Ziphias cavirostris 7:129

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CONTENTS

Biological Studies of the Neuse River Waterdog,

Necturus lewisi (Brimley), an Endemic North Carolina

Salamander (Amphibia: Proteidae)

The Necturus lewisi Study: Introduction, Selected Literature Review,

and Comments on the Hydrologic Units and Their Faunas. John E.

Cooper and Ray E. Ashton, Jr 1

Distribution, Ecology, and Feeding Habits of Necturus lewisi (Brimley).

Alvin L. Braswell and Ray E. Ashton, Jr 13

Chromosome Evolution in the Genus Necturus. Stanley K. Sessions and

John E. Wiley 37

The Testis and Reproduction in Male Necturus, with Emphasis on N.

lewisi (Brimley). Jeffrey Pudney, Jacob A. Canick and Gloria V.

Callard 53

Salamander Skin Toxins, with Special Reference to Necturus lewisi

(Brimley). Ronald A. Brandon and James E. Huheey 75

Field and Laboratory Observations on Microhabitat Selection, Move-

ments, and Home Range of Necturus lewisi (Brimley). Ray E.

Ashton, Jr 83

Pesticide and PCB Residues in Necturus lewisi (Brimley). Russell J.

Hall, Ray E. Ashton, Jr. and Richard M. Prouty 107

Miscellany Ill

Table of Contents, Nos. 7 and 8 (1982) 112

Index to Scientific Names, Nos. 7 and 8 (1982) 115