Seasonal and Tidally Driven Reproductive Patterns in the Saltmarsh

Topminnow, Fundulus jenkinsi

Erik T. Lang1,2, Nancy J. Brown-Peterson1, Mark S. Peterson1, and William T. Slack3

Fundulus jenkinsi is recognized federally and within a number of northern Gulf of Mexico states as a Species of Concern.Little is known about its life history, but a detailed reproductive histology study of F. jenkinsi can provide the foundationneeded to quantify reproductive parameters in this rare species in need of conservation. Monthly gonadosomatic index(GSI) of male and female F. jenkinsi indicated a spawning season from April through August. However, ovarianhistological analysis suggested March through August was a more accurate spawning season. The multiple oocytestages within F. jenkinsi in the spawning capable reproductive phase indicates batch spawning, similar to othermembers of its family. Many estuarine members of the family Fundulidae exhibit a semilunar spawning pattern, yet theoocyte composition of ovaries of F. jenkinsi suggested spawns occur multiple days around the time of spring tides bothwithin a population and on the individual level. Spawning did not occur on neap tides, and no late secondary growthvitellogenic oocytes (SGl) were found in the majority of females captured during neap tide. The lack of SGl oocytes infemales during the spawning season suggests the necessity for establishing a new sub-phase within the spawningcapable phase, termed the redeveloping sub-phase. This new sub-phase is applicable to other batch spawning specieswith group synchronous oocyte development. This work contributes to a better understanding of the importance ofintertidal saltmarsh habitat to F. jenkinsi, as spawning intensity appears to increase with tidal height and marshinundation.

MANY fishes rely on environmental cues to stim-ulate reproduction. In fact, Taylor (1984) pointsout that fishes in both intertidal and reef habitats

use lunar cycles, tidal dynamics, and their own endogenouscycles as cues for spawning. Several atherinomorph speciesuse spring tides to deposit eggs in intertidal habitats wherethe eggs incubate in air (Thompson and Thompson, 1919;Clark, 1925; Middaugh, 1981; Martin et al., 2004). Thefamily Fundulidae, in the superorder Atherinomorpha,comprises a large group of freshwater and saltwaterkillifishes. Coastal killifish, much like grunion (Thompsonand Thompson, 1919; Martin et al., 2004), predominantlyuse the tides to reproduce in areas inundated during or closeto spring tide events, which allows the eggs to incubate inair, and to receive more oxygen, while providing protectionfrom predators (Taylor et al., 1977; Taylor, 1984; DeMartini,1999). Along the east coast, fundulid reproduction isthought to occur in a semilunar pattern according to themoon phase cycles, which is synchronized with thesemidiurnal tidal cycle. Fundulus on the east coast havebeen shown to spawn during a spring tide when there iseither a full or new moon (Taylor and DiMichele, 1980;Taylor, 1991; Hsiao et al., 1994). However, northern Gulf ofMexico (GOM) tides are diurnal and the tides coincide withthe moon declination rather than lunar phase (Greeley andMacGregor, 1983). Because spring tides do not always occuron full or new moons, it is unclear whether fundulids foundalong the coast of the northern GOM use the samesemilunar spawning pattern as the Mummichog, Fundulusheteroclitus, on the Atlantic coast.

The Saltmarsh Topminnow Fundulus jenkinsi has beenlisted as a NOAA Species of Concern since 2004 (69 FR19975), but recently its sole jurisdiction has been placedunder USFWS and is currently being considered for‘threatened’ status under the Endangered Species Act (76

FR 49412; 10 August 2011). This species inhabits Spartinaand Juncus saltmarshes of the northern GOM (Thompson,1999; Peterson et al., 2003; NOAA, 2009) and little is knownabout the reproductive life history of this rare killifish. Lopezet al. (2010) estimated from oocyte diameters that F. jenkinsispawn from spring through the summer; reproductivehistology is required for a complete understanding of thereproductive life history of F. jenkinsi.

The reproductive histology of F. grandis, the Gulf Killifish,is described as a semilunar reproductive cycle (Greeley et al.,1988). This species invests more energy in a spawning peakat the beginning of the spawning period due to an almostdaily spawning regime at the beginning of the season; amore semilunar pattern becomes evident towards the end ofthe season (Greeley et al., 1988). Other coastal killifishesthat spawn during a cluster of three to six consecutive daysduring spring tides are the Mummichog, F. heteroclitus fromthe Atlantic coast, and F. grandis and F. similis, the LongnoseKillifish, from the northern GOM (Greeley and MacGregor,1983; Greeley et al., 1986, 1988). The capability to spawndaily in captivity has been shown in F. heteroclitus, regardlessof the semilunar cycle (Shimizu, 1997). However, F.heteroclitus and F. grandis seemed to exhibit semilunarspawning in captivity (Hsiao and Meier, 1986, 1989).Petersen et al. (2010) observed a lack of semilunar spawningin F. heteroclitus from a population in Maine, whereas Taylor(1991) observed semilunar spawning periods without tidalinfluence in F. heteroclitus from Delaware Bay. Kneib andStiven (1978) also observed a bimodal peak in spawning in F.heteroclitus with a large peak between March and May and asmall peak in July. These inconsistencies may indicate adifference in reproductive behavior among various wildpopulations. In wild conditions, F. grandis, F. similis, F.heteroclitus, F. luciae (Spotfin Killifish), and Adinia xenica(Diamond Killifish) have exhibited spawning seasons from

1 Department of Coastal Sciences, University of Southern Mississippi, 703 East Beach Drive, Ocean Springs, Mississippi; E-mail: (ETL)erik.lang@noaa.gov; (NJBP) nancy.brown-peterson@usm.edu; and (MSP) mark.peterson@usm.edu. Send reprint requests to ETL.

2 Present address: NOAA, 3500 Delwood Beach Road, Panama City, Florida 32408.3 U.S. Army Engineer Research and Development Center, Waterways Experiment Station EE-A, 3909 Halls Ferry Road, Vicksburg, Mississippi

39180-6199; E-mail: todd.slack@usace.army.mil.Submitted: 7 December 2010. Accepted: 31 March 2012. Associate Editor: K. Martin.F 2012 by the American Society of Ichthyologists and Herpetologists DOI: 10.1643/CP-10-187

Copeia 2012, No. 3, 451–459

mid-March to August or mid-September (Hastings andYerger, 1971; Kneib, 1978; Kneib and Stiven, 1978; Greeleyand MacGregor, 1983; Greeley et al., 1986).

A description of the reproductive life history of F. jenkinsiis vital for a better understanding of this rare species thatrequires conservation measures. Thus, the specific objectivesof this study are to: 1) quantify the spawning season in F.jenkinsi using the gonadosomatic index; 2) describe ovariandevelopment of F. jenkinsi with detailed reproductivehistology; and 3) determine whether spawning of F. jenkinsiis cued by tides or lunar phases.

MATERIALS AND METHODS

Seasonal collections.—Fundulus jenkinsi (Fig. 1) were col-lected in the Pascagoula River (30.39uN, 88.63uW; threesites, 295 fish), Grand Bay National Estuarine ResearchReserve (NERR; 30.39uN, 88.44uW; one site, 160 fish),Simmons Bayou (30.38uN, 88.76uW; one site, 13 fish),Davis Bayou (30.40uN, 88.77uW; one site, 60 fish), and FortBayou (30.42uN, 88.79uW; one site, 83 fish) along theMississippi coast during spring tides from April throughOctober 2008 and February through March of 2009.Multiple haphazardly chosen sites were sampled and allcollections were obtained within a five-day period from twodays before to two days after the spring tide monthly usingstandard minnow traps baited with Blue Crab (Callinectessapidus). Spring tide dates and times at each site were

obtained from the www.freetidetables.com website. Fishwere collected leaving the flooded marsh as the tidereceded and were immediately fixed in 10% neutralbuffered formalin (NBF). The wet weight (WW, mg), totallength (TL, mm), and gonad weight (GW, mg) weremeasured for each fish, and entire ovaries were placed intoindividually labeled plastic cassettes and reimmersed in10% NBF for reproductive histology analysis.

Semilunar collections.—To determine spawning frequency forthe two-week period between spring tides, sampling wasconducted between 14, 16, 18, 21, and 25 June 2010 in asection of Bayou Cumbest (30.39uN, 88.44uW) in Grand BayNERR. Standard minnow traps were used for collection andall fish were fixed in 10% NBF as above. The predicted hightide level of each day for the 2010 samples was taken fromtide charts (www.freetidetables.com) for Point Aux Pinesboat launch (30.38uN, 88.44uW), which is about one mileeast of the collection site.

Histological procedures.—Cassettes containing the entireformalin-fixed ovary were rinsed overnight in running tapwater, and tissue was dehydrated in graded ethanols, clearedin xylene substitute, and embedded in paraffin. Someovaries were lost due to tissue processor malfunctions andwere not able to be examined. All other ovaries weresectioned at 4 mm, mounted onto slides, and stained usingHematoxylin 2 (Richard Allen) and Eosin-Y (Richard Allen).Ovarian tissue was classified into reproductive phasesfollowing Brown-Peterson et al. (2011) and oocyte termi-nology was adapted from Grier et al. (2009). Representativeoocytes of each stage were measured (mm) from thehistological slides. The percentages of each oocyte stagewere determined for semilunar collections. Ovarian sectionson each slide were photographed using a Jenopik ProgResC5 dissection microscope camera and loaded into ProgResCapture 2.5 software. A microscope micrometer was used tocalibrate pixels into 1 mm measurements for proper areaestimation. The ovarian tissues were traced and the area(mm2) was obtained for each oocyte stage to the nearest 1 3

1025 mm2. The total area of the ovary was the summation ofthe areas for all oocyte stages; empty spaces between oocyteswere not included in these calculations. The area of eachoocyte stage was expressed as a percentage of the total areaof the ovary and the percentages were then averaged perdate.

Fig. 1. Images of a 50.0 mm TL male (top) and 53.6 mm TL female(bottom) Fundulus jenkinsi. Images taken by Gretchen L. Grammer.

Table 1. Number and Total Length (mm, Mean ± SE) and Range of Fundulus jenkinsi Collected by Month.

Date

Female Male

n

Total length (mm)

n

Total length (mm)

Mean ± SE Min–Max Mean ± SE Min–Max

February 56 44.0 6 1.01 26.6–65.8 15 36.0 6 1.44 29.0–48.1March 45 41.5 6 1.42 28.3–51.4 27 35.6 6 1.16 25.3–53.5April 81 45.6 6 0.58 35.6–69.6 16 41.7 6 1.22 30.0–52.3May 95 49.4 6 0.45 42.4–66.4 24 46.8 6 1.14 40.0–60.3June 45 51.4 6 0.72 42.5–70.3 18 46.6 6 1.10 40.8–59.9July 28 53.0 6 0.67 48.1–63.7 11 46.0 6 1.05 41.4–52.0August 45 50.9 6 0.65 43.3–61.0 40 47.5 6 0.56 34.0–52.7September 25 48.3 6 0.85 40.7–54.3 17 45.7 6 0.65 37.9–48.7October 15 44.8 6 1.78 29.7–56.5 8 44.6 6 0.91 41.4–49.2

452 Copeia 2012, No. 3

Analysis.—The gonadosomatic index (GSI) was used toestimate spawning preparedness, where GSI 5 [GW/(WW2GW)] 3 100. All GSI data were arcsin square-roottransformed prior to analysis. Linear regression of arcsinsquare-root transformed GSI values versus gonad-free bodyweight (GFBW) was conducted separately for males andfemales to determine if the GSI was influenced by GFBW. Aone-way ANOVA was used to determine if there was asignificant difference among monthly mean GSI values. If asignificant F-value was calculated, a post-hoc Sidak testfollowed. Homogeneity of variance (Levene’s statistic) andnormality of distribution (Kolmogorov-Smirnov test) weretested prior to analysis. If arcsin square-root transformed GSIdata proved to be heterogeneous, a Games-Howell (GH) testwas used instead of a Sidak for pairwise comparisons. AllP-values # 0.05 were considered significant and statisticswere processed using SPSS software (ver. 15).

RESULTS

Seasonal collections.—A total of 435 female and 176 male F.jenkinsi were collected during the course of the study, withthe largest female reaching 70.3 mm TL and the largest malereaching 60.3 mm TL (Table 1). Male body weight was notpredictive of GSI (r2 5 0.018, P 5 0.074). Female bodyweight was predictive of GSI values (r2 5 0.074, P , 0.0001),yet the relationship explained only 7.4% of the variance inGSI and was considered biologically insignificant. Trans-formed GSI values were heteroscedastic (P , 0.05) but werenormally distributed (P . 0.05).

Mean female GSI among months was significantlydifferent (ANOVA: F8,426 5 88.018, P , 0.0001), with valuesfrom April through August higher than the other months(GH; P , 0.05; Fig. 2). March GSI values were significantlyhigher than those in February, September and October.Mean male GSI was also significantly different amongmonths (ANOVA: F8,167 5 18.636, P , 0.0001), with valuesfrom February through August significantly higher thanthose in September and October (GH; P . 0.05; Fig. 2).Overall, GSI values suggest an April through Augustspawning season in Mississippi.

Histological analysis revealed that in February all femaleswere in the developing phase (Table 2), characterized in F.jenkinsi by the presence of primary growth (PG; ,0.25 mm

Fig. 2. Plot of mean (6SE) female and male gonadosomatic index(GSI) by month for Fundulus jenkinsi. In October, SE bars were so smallthey were hidden by the data point symbol. Data points with the sameletters within gender are not significantly different (P . 0.05).

Tab

le2.

Repro

du

ctiv

ePh

ase

Perc

en

tages

of

Fun

dulu

sje

nki

nsi

by

Mo

nth

Use

dto

Dete

rmin

eSp

aw

nin

gSeaso

n.Sam

ple

size

an

dth

em

in–

max

TL(m

m)

valu

es

are

pro

vid

ed

inp

are

nth

ese

s.

Sp

aw

nin

gp

hase

(%)

Mo

nth

of

cap

ture

Feb

ruary

(n=

4)

(42

.5–

64

.2)

Marc

h(n

=1

6)

(28

.3–

51

.5)

Ap

ril

(n=

33

)(4

0.8

–6

2.6

)M

ay

(n=

72

)(4

2.4

–6

2.4

)Ju

ne

(n=

38

)(4

2.5

–7

0.3

)Ju

ly(n

=1

7)

(48

.1–

63

.7)

Au

gu

st(n

=1

6)

(44

.9–

60

.6)

Sep

tem

ber

(n=

8)

(48

.4–

54

.3)

Oct

ober

(n=

9)

(29

.7–

56

.5)

Dev

elopin

g1

00

25

00

00

00

0Sp

awnin

gca

pab

le0

50

35

61

45

59

56

25

0Act

ivel

ysp

awnin

g0

25

65

39

55

41

44

00

Reg

ress

ing

00

00

00

07

54

4Reg

ener

atin

g0

00

00

00

05

6

Lang et al.—Saltmarsh Topminnow reproduction 453

diameter), cortical alveolar (CA; 0.25–0.50 mm), and earlysecondary growth (SGe; .0.50–0.70 mm) oocytes (Fig. 3A).From March through September, F. jenkinsi entered into thespawning capable phase as denoted by the appearance oflate secondary growth (SGl; .0.7–1.0 mm) and fully grown(SGfg; .1.0–1.3 mm) oocytes (Fig. 3B; Table 2). The pres-ence of oocytes in multiple developmental stages, as well as

the presence of postovulatory follicles (POF; Fig. 3B, 3C) inthe spawning capable phase, provides evidence that F.jenkinsi is a batch spawner. The actively spawning sub-phaseof the spawning capable phase indicates spawning isimminent, and is characterized by oocytes undergoingoocyte maturation (OM, .1.3 mm; Fig. 3C). Eighty-eightpercent of actively spawning females showed OM in the

Fig. 3. Photographs illustrating spawning phases of female Fundulus jenkinsi. (A) Developing in March, (B) spawning capable in June, (C) activelyspawning in May, (D) regressing in September, and (E) regenerating in October. Oocyte stages are labeled as primary growth (PG), cortical alveoli (CA),early secondary growth (SGe), late secondary growth (SGl), full secondary growth (SGfg), oocyte maturation (OM), postovulatory follicles (POF), and atresia.

454 Copeia 2012, No. 3

same ovary that contained 12, 24, and/or 48 hr POF,indicating that an individual can spawn during severalconsecutive days (Fig. 4). Actively spawning females of F.jenkinsi were found each day in collections from 18–21 May2008, providing further evidence that, on the populationlevel, this species is capable of spawning during severalconsecutive days in the wild. Twenty-five percent of femaleshistologically examined in March were in the activelyspawning sub-phase, and a high percentage of activelyspawning females were captured from April through August(Table 2). All histologically examined females during Aprilthrough August were in the spawning capable phase, whichalso includes the actively spawning sub-phase.

By September, 75% of the females had completed thespawning season and entered the regressing phase, charac-terized by atresia of vitellogenic oocytes and occasional CAoocytes (Fig. 3D). The majority of females were in theregenerating phase in October (Fig. 3E, Table 2), histologi-cally characterized by domination of PG oocytes, withoccasional CA oocytes and late stage atresia present in someindividuals.

Although the female GSI values indicated that spawningstarted in April, the reproductive histology showed fish inthe actively spawning sub-phase in March. However, thelack of OM and POFs in September indicates the spawningseason appears to end in August, similar to conclusionsdrawn from the GSI data. Therefore, F. jenkinsi appears tospawn from March through August in Mississippi watersbased on histological analysis.

Semilunar collection.—A total of 58 females were collectedbetween 14, 16, 18, 21, and 25 June 2010 between twospring tides to investigate tidally influenced spawningperiodicity. Fish ranged in size from 41.2 to 64.6 mm TL(Table 3). Gonad-free body weight of females collected from14 to 25 June 2010 was not predictive of arcsin square-roottransformed GSI values (r2 5 0.025, P 5 0.233). Transformedfemale GSI values were normally distributed (P . 0.05), butwere heteroscedastic (P , 0.05). The transformed GSI valuesvaried over the tidal cycle (ANOVA: F4,53 5 18.164, P ,

0.0001), with higher values on spring tide and lower values

during neap tide (Fig. 5). The GH test showed there was nodifference between 16, 18, and 21 June 2010 GSI values (GH:P . 0.05), and 14 and 21 June 2010 were also not different(GH: P . 0.05). The 25 June 2010 mean GSI value wasgreater than all other GSI values (Fig. 5), suggesting GSIvalues were significantly higher during the peak spring tidesand indicating spawning occurs during spring tide but notduring neap tide.

Oocyte stages were also examined from spring tide tosuccessive spring tide. This analysis indicated that, evenwithin the spawning season, the ovaries of fish during neaptide consisted mainly of SGe oocytes and there was a highpercentage of atretic oocytes during the neap tide period(Table 4), although this was not significant (ANOVA: F4,515

1.113, P 5 0.360). Furthermore, SGfg oocytes were rare,occurring in three of the 15 fish collected on that date, andno oocytes undergoing OM were present on the neap tide(Table 4). However, oocytes undergoing OM were presenton the days after and before the neap tide period (Table 4,Fig. 6). The presence of OM but lack of POFs on 21 June2010 indicates that spawning resumed on that date after atleast a 48 h hiatus during the neap tide on 18 June 2010.This suggests that, within this particular population,spawning occurs on and around the spring tide, and ceasesduring the neap tide period.

Some females entered a spawning phase just before andduring neap tide that resembles the developing phase, basedon the lack of SGl and SGfg oocytes present in the ovary.However, these fish should still be considered spawningcapable, since they have likely spawned previously duringthe six-month spawning season. Therefore, we have estab-lished a new sub-phase within the spawning capable phase,termed the redeveloping sub-phase, to more accuratelyclassify these fish (Table 5). While there are no specifichistological markers to distinguish this new redevelopingsub-phase, characteristics of this sub-phase for females batchspawning include fish captured during an extended spawn-ing season with ovaries containing SGe oocytes, but with noevidence of SGl or SGfg oocytes and/or POF # 48 h.

Peak spawning occurs on spring tide as indicated by thehigh percentage of females in the actively spawning sub-phase, and no spawning takes place on a neap tide (Fig. 6).Furthermore, it appears that the most intense spawningmay be cued by tidal height. When the tide was above60 cm, the percent area of the ovary occupied by oocytesundergoing OM was above 15%, but this percentagedecreased when the tide fell below the 60 cm mark(Fig. 6). Some actively spawning females are also foundseveral days before and after the spring tide (Table 5). Thenewly defined redeveloping sub-phase was only foundimmediately preceding and during neap tide, although

Fig. 4. Image of an ovarian histological section of Fundulus jenkinsi(52.3 mm TL) in May in the actively spawning sub-phase showingvarious stages of postovulatory follicles (POF) as well as oocytematuration (OM).

Table 3. Number and Total Length (mm, Mean ± SE) and Range ofFemale Fundulus jenkinsi by Date for Fish Captured between TwoSpring Tides.

Date n

Total length (mm)

Mean ± SE Min–Max

6/14/2010 17 50.3 6 1.18 43.6–64.66/16/2010 7 51.8 6 0.78 48.1–53.56/18/2010 15 50.7 6 1.26 44.5–60.56/21/2010 6 52.1 6 0.66 50.7–54.16/25/2010 13 47.6 6 1.64 41.2–64.0

Lang et al.—Saltmarsh Topminnow reproduction 455

some females captured during these dates were still inthe spawning capable phase and contained SGl oocytes(Table 5). Thus, both the histological analysis and the GSIvalues suggest F. jenkinsi spawns on days around the springtide but not during the neap tide. Furthermore, individualfemales appear capable of spawning several times in thedays around the spring tides, as evidenced by the co-occurrence of oocytes undergoing OM and POF # 48 h inthe ovary (Figs. 4, 6).

DISCUSSION

Fundulus jenkinsi is a patchily distributed species throughoutits range (see Lopez et al., 2011 for distribution map ofknown range), and, until recently, few data were availableon its life history and ecology (Peterson et al., 2003; Lopez etal., 2010, 2011). Boschung and Mayden (2004) providedetailed drawings of adults, whereas Lang et al. (2011)provide detailed illustrations of individuals ,15 mm TL.Lopez et al. (2010) illustrates important dimorphic diagnos-tic characters.

It has become common knowledge that GSI alone cannotaccurately determine spawning readiness, and reproductivehistology provides direct biological information that con-firms or refines the GSI results (Jons and Miranda, 1997). For

example, GSI values of female F. jenkinsi suggest an Aprilthrough August spawning season, while ovarian histologicalanalysis shows the season begins in March. However, theend of the spawning season in August is consistent betweenGSI and histology. Therefore, we consider the spawningseason of F. jenkinsi to be March–August in Mississippi, butthis conflicts with Lopez et al. (2010) who suggested about35% of F. jenkinsi collected in the winter were spawningcapable based on oocyte diameters. However, the month ofMarch was included as winter in Lopez et al. (2010). Male F.jenkinsi exhibited spawning coloration before and after thespawning season, and GSIs indicated males were spawningcapable in February, although females were not yet preparedfor reproduction. Therefore, the beginning of the spawningseason cannot completely be assumed by the appearance ofmale sexual coloration.

The spawning season of F. jenkinsi coincides with thespawning seasons of F. grandis, F. similis, and A. xenica fromthe GOM (Gunter, 1945; Hastings and Yerger, 1971; Greeleyand MacGregor, 1983; Greeley et al., 1986). A west coastfundulid, F. parvipinnis (California killifish), spawns fromApril through September (Fritz, 1975), and, on the eastcoast, F. heteroclitus and F. luciae spawn from March throughAugust (Byrne, 1978; Kneib, 1978; Kneib and Stiven, 1978).All estuarine fundulids seem to be spring to summer batchspawners that use high tides to deposit their eggs in a securemarsh environment. Although the dip in GSI for F. jenkinsishown in June was not significantly lower than the rest ofthe spawning season GSI values, other fundulids haveexhibited more exaggerated bimodal spawning peaks (Kneiband Stiven, 1978). Harrington (1959) suggested the mid-summer pause in spawning of F. confluentus (Marsh Killifish)may be due to a high temperature threshold in the shallow,intertidal waters of Florida.

The histological sections of ovaries of F. jenkinsi show thelarge oocytes common in fundulids that are characteristic ofdemersal spawning fishes. Typically, fecundity of demersalspawners is less than that of pelagic species that spawnmuch smaller oocytes. Unfortunately, there are no dataavailable from this study or from the literature indicatingthe batch size of F. jenkinsi. Individual F. jenkinsi appear tospawn several times around spring tides. Not only is OMdisplayed on numerous days between spring tides, but POFsare also indicative of spawning during those days. Spawningof the population is estimated to occur during about nine ofthe 14 days between spring tides. Tide height and inundatedmarsh are used by F. jenkinsi as spawning indicators, and

Fig. 5. Plot of mean (6SE) female gonadosomatic index (GSI) ofFundulus jenkinsi between two spring high tides (cm) by date. Datapoints with the same letters are not significantly different (P . 0.05).Moon phases included above graph on corresponding dates.

Table 4. Mean Oocyte Stage Percentages (±SE) of Fundulus jenkinsi by Date for Fish Captured between Two Spring Tides.

Oocyte stage (%)

Tidal stage and date of capture

Spring6/14/2010(n = 16)

Descending6/16/2010

(n = 7)

Neap6/18/2010(n = 15)

Ascending6/21/2010

(n = 6)

Spring6/25/2010(n = 13)

Primary growth 4.0 6 0.5 7.1 6 0.9 6.6 6 0.9 2.3 6 0.9 1.9 6 0.4Cortical alveoli 3.5 6 0.7 3.8 6 1.0 4.9 6 0.6 1.3 6 0.8 1.7 6 0.4Early secondary growth 25.0 6 2.4 46.2 6 7.6 66.0 6 6.5 49.1 6 5.4 22.6 6 2.3Late secondary growth 11.7 6 3.1 22.7 6 9.0 11.5 6 5.5 28.7 6 3.8 26.7 6 4.4Full grown 27.2 6 4.1 10.2 6 6.9 4.4 6 2.3 13.5 6 4.7 25.7 6 5.0Oocyte maturation 20.1 6 5.3 3.5 6 3.5 0.0 6 0.0 1.7 6 1.7 17.5 6 5.1Post-ovulatory follicles 7.8 6 1.1 3.6 6 0.9 0.0 6 0.0 0.0 6 0.0 2.2 6 0.4Atresia 0.7 6 0.2 2.8 6 0.6 6.7 6 1.3 3.2 6 1.5 1.6 6 0.5

456 Copeia 2012, No. 3

spawning intensity appears to increase with tide heightregardless of the decoupling of spring tides with new andfull moons. For instance, while spring tides closely corre-sponded with new and full moon dates in June 2010, therewas a difference of around three to five days between springtide and new/full moon in March 2009 and April 2008(www.freetidetables.com). The ability of F. jenkinsi to spawnthrough a cluster of days is similar to populations ofF. grandis from Alabama (Greeley and MacGregor, 1983;Greeley et al., 1988). However, spawning of F. grandis and F.similis was reported to only occur on late ascending tidesand spring tides (Greeley and MacGregor, 1983; Greeleyet al., 1986), while some F. jenkinsi also spawned on earlydescending tides. Despite the ability of F. grandis and F.similis to spawn on ascending high tide in addition to springtides, Greeley et al. (1986, 1988) contended that bothspecies spawned in synchrony with moon declination andspring tides during the middle and late months of thespawning season, and therefore were semilunar spawners. Incontrast, Waas and Strawn (1983) contended that there wasa ‘‘lack of lunar effect’’ on the spawning of F. grandis inGalveston Bay, Texas and attributed the lack of lunarsynchronization to wind overriding the lunar tides. Petersenet al. (2010) showed a lack of semilunar spawning in F.heteroclitus and attributed the behavior to the shorterspawning season due to the boreal climate of Maine. Thesemilunar spawning of F. heteroclitus in Delaware indicatesthat spawning of this species may vary along the east coast(Taylor and DiMichele, 1980; Taylor, 1984; Hsiao et al.,1994). The wind-driven tides and small tidal differences of

the northern GOM coast lead to marsh inundation periodsof longer duration but aperiodic occurrence compared to theAtlantic coast, which may have resulted in an adaptation forspawning on consecutive days in GOM species (Waas andStrawn, 1983; Kneib, 1997). The propensity of F. jenkinsi tospawn on descending tides in addition to the ascending andspring tides may be due to its shallow water depthpreference (Peterson et al., 2003; Lopez et al., 2011).

Some female F. jenkinsi had no late secondary growthoocytes or full grown oocytes before and during neap tide,similar to fish found in February in the developing phase.Because fish in this condition were present in the middleof the spawning season and fish cannot return to thedeveloping phase from the spawning capable phase (Brown-Peterson et al., 2011), this ovarian condition was character-ized as a new sub-phase of spawning capable calledredeveloping. A similar situation was documented in Sardinapilchardus (European Pilchard) in which 39% of maturefemales exhibited this redeveloping sub-phase in peakspawning months (Konstantinos Ganias, Aristotle Universi-ty of Thessaloniki, Greece, pers. comm.). The redevelopingsub-phase and lack of SGl and SGfg oocytes during thespawning season may be common in other batch spawningspecies that use lunar or tidal cues for spawning or have along (10–13 day) interspawning interval (i.e., Ganias et al.,2004). In contrast, SCl and SGfg oocytes are always presentduring the spawning season in many other batch spawningspecies (Lowerre-Barbieri et al., 2011).

Detailed reproductive histology determines when a fishcould spawn or has already spawned, which is valuable interms of estimating reproductive condition at the individuallevel. The presence of multiple oocyte stages not onlysupports asynchronous oocyte development of F. jenkinsi,but also demonstrates the ability of a single individual of F.jenkinsi to spawn over successive days. Other members of thefamily Fundulidae (F. grandis and F. heteroclitus) are knownto spawn multiple days in a row as a population (Waas andStrawn, 1983; Greeley et al., 1988; Petersen et al., 2010), butamong fundulids, only in F. heteroclitus has a previous reportshown an individual capable of spawning daily (Shimizu,1997). In contrast, our results indicate that F. jenkinsi spawnsover successive days in the wild, with ovarian histologyshowing OM along with 12, 24, and/or 48 hr POFs present.This pattern is repeated in more than 88% of the activelyspawning fish from April through August, suggesting thisovarian condition is common in F. jenkinsi and the majorityof the individuals in this Mississippi population spawnmultiple days in a row throughout the reproductive season.The ability of an individual F. jenkinsi to spawn for up to fourdays successively sheds new light on the reproductivestrategy of the species and also adds information necessaryfor population sustainability estimates.

Fig. 6. Percentage of the ovaries of Fundulus jenkinsi capturedbetween two spring tides containing oocytes undergoing oocytematuration and various stages of postovulatory follicles (POF). Moonphases included above graph on corresponding dates.

Table 5. Spawning Phase Percentages of Fundulus jenkinsi by Date for Fish Captured between Two Spring Tides.

Spawning phase (%)

Tidal stage and date of capture

Spring6/14/2010(n = 16)

Descending6/16/2010

(n = 7)

Neap6/18/2010(n = 15)

Ascending6/21/2010

(n = 6)

Spring6/25/2010(n = 13)

Spawning capable 44 57 47 83 38Actively spawning 56 14 0 17 62Redeveloping 0 29 53 0 0

Lang et al.—Saltmarsh Topminnow reproduction 457

The quantification of the reproductive strategy of this rarefundulid provides additional data concerning the linkage oflife history with critical salt marsh habitat. Fundulus jenkinsiuses high tides to invade the high marsh where eggs arestranded to incubate in air throughout the reproductiveseason. According to Lopez et al. (2010, 2011), upper marsh isa key habitat for F. jenkinsi, and wetland alteration anddegradation throughout its range would significantly impactthe feeding and breeding habitat of this small fundulid(Peterson and Lowe, 2009).

ACKNOWLEDGMENTS

We thank the USFWS in conjunction with the MississippiMuseum of Natural Science under the Mississippi Depart-ment of Wildlife Fisheries and Parks for funding this project.We also thank J. Lopez, P. Grammer, M. Lowe, J. Havrylkoff,and M. Andres for field assistance, and G. Grammer forimages in Fig. 1. This research was conducted with a blanketsampling permit to the USM-Gulf Coast Research Laboratoryand was approved and performed under the USM IACUCprotocol #10040802.

LITERATURE CITED

Boschung, H. T., Jr., and R. L. Mayden. 2004. Fishes ofAlabama. Smithsonian Books, Washington, D.C.

Brown-Peterson, N. J., D. M. Wyanski, F. Saborido-Rey,B. J. Macewicz, and S. K. Lowerre-Barbieri. 2011. Astandardized terminology for describing reproductivedevelopment in fishes. Marine and Coastal Fisheries 3:52–70.

Byrne, D. M. 1978. Life history of the spotfin killifish,Fundulus luciae (Pisces: Cyprinodontidae), in Fox Creekmarsh, Virginia. Estuaries 1:211–227.

Clark, F. N. 1925. The life history of Leuresthes tenuis, anatherine with tide controlled spawning habits. CaliforniaDepartment of Fish and Game Fisheries Bulletin 10:1–51.

DeMartini, E. E. 1999. Intertidal spawning, p. 143–164. In:Intertidal Fishes: Life in Two Worlds. M. H. Horn, K. L. M.Martin, and M. A. Chotkowski (eds.). Academic Press, SanDiego, California.

Fritz, E. S. 1975. The life history of the California killifishFundulus parvipinnis Girard in Anaheim Bay, California,p. 91–106. In: The Marine Resources of Anaheim Bay. E. D.Lane and C. W. Hill (eds.). Fish Bulletin 165, CaliforniaDepartment of Fish and Game, Sacramento, California.

Ganias, K., S. Somarakis, A. Machias, and A. Theodorou.2004. Pattern of oocyte development and batch fecundityin the Mediterranean sardine. Fisheries Research 67:13–23.

Greeley, M. S., Jr., and R. MacGregor. 1983. Annual andsemilunar reproductive cycles of the gulf killifish, Fundu-lus grandis, on the Alabama Gulf Coast. Copeia 1983:711–718.

Greeley, M. S., Jr., R. MacGregor, and K. R. Marion. 1988.Changes in the ovary of the Gulf killifish, Fundulus grandis(Baird and Girard), during seasonal and semilunar spawn-ing cycles. Journal of Fish Biology 1988:97–107.

Greeley, M. S., Jr., K. R. Marion, and R. MacGregor. 1986.Semilunar spawning cycles of Fundulus similis (Cyprino-dontidae). Environmental Biology of Fishes 17:125–131.

Grier, H. J., M. C. Uribe-Aranzabel, and R. Patino. 2009. Theovary, folliculogenesis, and oogenesis in teleosts, p. 25–84.In: Reproductive Biology and Phylogeny of Fishes (Ag-

nathans and Bony Fishes). Volume 8A. B. G. M. Jamieson(ed.). Science Publishers, Enfield, New Hampshire.

Gunter, G. 1945. Studies on the marine fishes of Texas.Publications of the Institute of Marine Science, Universityof Texas 1:1–190.

Harrington, R. W., Jr. 1959. Delayed hatching in strandedeggs of marsh killifish, Fundulus confluentus. Ecology 40:430–437.

Hastings, R. W., and R. W. Yerger. 1971. Ecology and lifehistory of the diamond killifish, Adinia xenica (Jordan andGilbert). American Midland Naturalist 86:276–291.

Hsiao, S-M., M. S. Greeley, Jr., and R. A. Wallace. 1994.Reproductive cycling in female Fundulus heteroclitus.Biological Bulletin 186:271–284.

Hsiao, S-M., and A. H. Meier. 1986. Spawning cycles of theGulf killifish, Fundulus grandis, in closed circulationsystems. Journal of Experimental Zoology 240:105–112.

Hsiao, S-M., and A. H. Meier. 1989. Comparison ofsemilunar cycles of spawning activity in Fundulus grandisand F. heteroclitus held under constant laboratory condi-tions. Journal of Experimental Zoology 252:213–218.

Jons, D., and E. Miranda. 1997. Ovarian weight as an indexof fecundity, maturity, and spawning periodicity. Journalof Fish Biology 50:150–156.

Kneib, R. T. 1978. Habitat, diet, reproduction and growth ofthe spotfin killifish, Fundulus luciae, from a North Carolinasalt marsh. Copeia 1978:164–168.

Kneib, R. T. 1997. The role of tidal marshes in the ecology ofestuarine nekton. Oceanography and Marine Biology:Annual Review 35:163–220.

Kneib, R. T., and A. E. Stiven. 1978. Growth, reproduction,and feeding of Fundulus heteroclitus (L.) on a NorthCarolina salt marsh. Journal of Experimental MarineBiology and Ecology 31:121–140.

Lang, E. T., M. S. Peterson, and W. T. Slack. 2011.Comparative development of five sympatric coastalfundulids from the northern Gulf of Mexico. Zootaxa2901:1–18.

Lopez, J. D., M. S. Peterson, E. T. Lang, and A. M.Charbonnet. 2010. Linking habitat and life history forconservation of the rare saltmarsh topminnow Fundulusjenkinsi: morphometrics, reproduction, and trophic ecol-ogy. Endangered Species Research 12:141–155.

Lopez, J. D., M. S. Peterson, J. Walker, G. L. Grammer, andM. S. Woodrey. 2011. Distribution, abundance, andhabitat characterization of the saltmarsh topminnow,Fundulus jenkinsi (Everman 1892). Estuaries and Coasts34:148–158.

Lowerre-Barbieri, S. K., K. Ganias, F. Saborido-Rey, H.Murua, and J. R. Hunter. 2011. Reproductive timing inmarine fishes: variability, temporal scales, and methods.Marine and Coastal Fisheries 3:71–91.

Luna, L. G. 1960. Manual of Histologic Staining Methods ofthe Armed Forces Institute of Pathology. Third edition.McGraw Hill Inc., Washington, D.C.

Martin, K. L. M., R. C. Van Winkle, J. E. Drais, and H.Lakisic. 2004. Beach-spawning fishes, terrestrial eggs, andair breathing. Journal of Physiological Biochemical Zool-ogy 77:750–759.

Middaugh, D. P. 1981. Reproductive ecology and spawningperiodicity of the Atlantic silverside, Menidia menidia(Pisces, Atherinidae). Copeia 1981:766–776.

National Oceanic and Atmospheric Administration(NOAA). 2009. Proactive conservation program: species

458 Copeia 2012, No. 3

of concern. NOAA Fisheries, Office of Protected Resources.www.nmfs.noaa.gov/pr/species/concern (6 October 2010).

Petersen, C. W., S. Salinas, R. L. Preston, and G. W.Kidder, III. 2010. Spawning periodicity and reproductivebehavior of Fundulus heteroclitus in a New England saltmarsh. Copeia 2010:203–210.

Peterson, M. S., G. L. Fulling, and C. M. Woodley. 2003.Status and habitat characteristics of the saltmarshtopminnow, Fundulus jenkinsi (Everman) in eastern Mis-sissippi and western Alabama coastal bayous. Gulf andCaribbean Research 15:51–59.

Peterson, M. S., and M. R. Lowe. 2009. Implications ofcumulative impacts to estuarine and marine fish andinvertebrate resources. Reviews in Fishery Science 17:505–523.

Shimizu, A. 1997. Reproductive cycles in a reared strain ofthe mummichog, a daily spawner. Journal of Fish Biology51:724–737.

Taylor, M. H. 1984. Lunar synchronization of fish repro-duction. Transactions of the American Fisheries Society113:484–493.

Taylor, M. H. 1991. Entrainment of the semilunar cycle ofFundulus heteroclitus. Reproductive Physiology of Fish91:157–159.

Taylor, M. H., and L. DiMichele. 1980. Ovarian changesduring the lunar spawning cycle of Fundulus heteroclitus.Copeia 1980:118–125.

Taylor, M. H., L. DiMichele, and G. J. Leach. 1977. Eggstranding in the life cycle of the mummichog, Fundulusheteroclitus. Copeia 1977:397–399.

Thompson, B. A. 1999. An evaluation of the saltmarshtopminnow, Fundulus jenkinsi. Final Report to the U.S.Department of Commerce, National Marine FisheriesService, SE Regional Office, St. Petersburg, Florida.

Thompson, W. F., and J. B. Thompson. 1919. Thespawning of the grunion (Leuresthes tenuis). CaliforniaDepartment of Fish and Game Fish Bulletin 3:1–29.

Waas, B. P., and K. Strawn. 1983. Seasonal and lunar cyclesin gonadosomatic indices and spawning readiness ofFundulus grandis. Contributions in Marine Science 26:127–141.

Lang et al.—Saltmarsh Topminnow reproduction 459