Thomas C. Kane and George D. Brunner.
Geographic variation in the cave beetle, Neaphaenops tellkampfi (Coleoptera: Carabidae).
Psyche 93:231-252, 1986.

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GEOGRAPHIC VARIATION IN THE CAVE BEETLE
NEAPHAENOPS TELLKAMPFI
(COLEOPTERA: CARABIDAE)
Department of Biological Sciences, University of Cincinnati Cincinnati. OH 45221 USA
More than 200 species of cave limited (i.e., troglobitic) trechine carabid beetles are known from caves of the eastern United States (Barr, 1979b, 1981). These species are generally considered to be derived from ancestral surface species which were widespread dur- ing the cold, moist climates associated with glacial maxima (Barr, 1968). Subsequent warming and drying of these regions, as glaciers retreated, led ultimately to the extirpation of surface populations, with only some of the cave limited stocks surviving. Available evi- dence suggests that for trechines cave isolation is irreversible (Barr, 1968, 1979a). Therefore, geographic spread of and gene flow in troglobitic trechines will be restricted to subterranean routes (Barr, 1968). The interconnectivity of caves and the presence of geological barriers (e.g., noncavernous strata and large rivers) become impor- tant factors in determining the geographic extent of and degrees of gene flow within these troglobitic taxa. In extensive and highly continuous limestone cave systems, such as those of the Mississippian plateaus, interpretation of evolution- ary relationships between closely similar taxa becomes especially complicated (Barr, 1979b). One question which arises is whether such taxa represent multiple isolations of a common surface dwell- ing ancestor or are the product of more recent divergence in a common troglobitic ancestor. Even when the latter scenario appears to be the case, divergence may only involve subtle, although gener- ally consistent, differences in minor morphological characters. Thus, inferences about such factors as the amount of gene flow, if 'Author to whom all editorial correspondence and reprint requests should be addressed.
Manuscript received by the editor March 25, 1986.



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232 Psyche [vo~. 93
any, still occurring among the taxa, the relative degree of differenti- ation between the various taxa, and the manner in which the present geographic pattern has been produced may be strengthened by the availability of genetic data such as those obtained through gel elec- trophoresis (Barr, 1979b; Turanchik and Kane, 1979). As Barr (1979b) has indicated, the large geographic distribution and abundance of Neaphaenops tellkampfi populations present an excellent opportunity to assess the extent of gene flow between local populations of a troglobitic trechine using both morphological and electrophoretic data. Among the many species of troglobitic tre- chine carabid beetles in the United States, Neaphaenops tellkampfi is noteworthy for having the most extensive geographic range and being one of the most abundant species of the group (Barr, 1979b, 1981). The species is distributed (Fig. 1) from just south of the Ohio River in the north to its southern limit near the Tennessee border, in the highly cavernous Mississippian limestones of the Pennyroyal Plateau in west central Kentucky (Barr, 1979b). The western extent of its range is delimited by the noncavernous Big Clifty sandstone, and the eastern and southeastern limits of the range correspond roughly with the contact with the Salem and Warsaw limestones (Barr, 1979b).
Neaphaenops tellkampfi, like other cave trechines, is an impor- tant predator in terrestrial cave communities (Barr and Kuehne, 1971; Kane and Poulson, 1976). Unlike other troglobitic trechines in the Pennyroyal Plateau, however, N. tellkampfi has evolved special- ized behaviors which allow it to prey on the eggs and early instar nymphs of the common cave "cricket" Hadenoecus subterraneus (Orthoptera: Rhaphidophoridae), resources which are energy rich and seasonally abundant (Kane and Poulson, 1976; Hubbell and Norton, 1978). This predator-prey interaction has evolved to the extent that no N. tellkampfi populations occur outside the range of H. subterraneus (Hubbell and Norton, 1978). In fact, Barr (1979b) has suggested that at least part of the eastern limits of the N. tell- kampfi range may be determined by the absence of H. subterraneus further east, rather than to the presence of any extrinsic geological barrier.
Using morphological and geological criteria, Barr (1979b) has recognized four subspecies of N. tellkampfi. The nominate subspe- cies, N. t. tellkampfi, on which most of the ecological studies dis-



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Kane & Brunner- Neaphaenops tellkampfi
KENTUCKY
HENROTI
V/A TOR
TELLKAMPFI
MER/DlONALIS
TELLKAMPFI X MERIDIONALIS
- St Louis / Salem and Warsaw Conlac'
Scale
0 10 20 30 40 mi
-
Figure 1. Map of west central Kentucky showing locations sampled for Nea- phaenops tellkampfi in this study. Taxonomic designations of populations at these sites (after Barr, 1979b) are as follows: N. t. henroti: BL; CW; SS; T; N. t. meridionalis: H; 0s; ST; N. t. tellkampfi: B; BH; GO; HA; LB; P; RB; WH; N. t. viator: C; CB; S; N. t. meridionalis X N. t. tellkampfi hybrid: F. cussed previously have been done, is distributed in the central portion of the range to include the caves of Mammoth Cave National Park. Neaphaenops t. meridionalis, the southern subspe- cies, is limited to the north by the noncavernous sandstones near the Barren River. However, two populations are known in the south- eastern part of the range which are morphologically intermediate between nominate tellkampfi and meridionalis for six of nine diag- nostic characters, suggesting a narrow zone of hybridization between the two subspecies. Barr (1979b) points out, however, that despite the limited gene flow, meridionalis is morphologically the most distinct of the four subspecies. Morphological evidence (Barr, 1979b) suggests a broad zone of hybridization between nominate tellkampfi and the eastern subspecies N. t. viator, with gradual intergradation between the two subspecies over approximately an



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234 Psyche [VOI. 93
eight km. distance. The eastern extent of the viator range is delim- ited by the contact of the St. Louis/Salem and Warsaw limestones and, perhaps more directly, by the absence of H. subterraneus further east (Barr, 1979b). As is the case with nominate tellkampfi, populations of viator are known from caves on both the north and south sides of the Green River. The northern limits of the viator range are set in large part by a sandstone ridge and extensive fault zone across Hart County. This geological feature also appears to be a complete barrier to gene flow between the northern subspecies N. t. henroti and either nominate tellkampfi or viator to the south (Fig. 1) (Barr, 1979b). Despite the absence of any known hybrid popula- tions, tellkampfi and henroti are the most similar subspecies morphologically, and henroti also shows a large degree of morpho- logical affinity with viator as well (Barr, 1979b). Previous studies using gel electrophoresis (Giuseffi et al., 1978; Turanchik and Kane, 1979) have shown that genetic variability in local populations of N. t. tellkampfi approach those observed in similar surface dwelling invertebrates. These results, coupled with similar subsequent findings in other species (e.g., Dickson et al., 1979), suggest that cave adaptation does not necessarily result in a reduction in genetic variation. Further, genetic similarity values (I) (Nei, 1972) among eight local populations of nominate tellkampfi fall in the range (i.e., 0.90-1 .OO (Turanchik and Kane, 1979) ) com- monly reported for populations of continuously distributed surface dwelling species. These results substantiate the contention that con- tinuous limestone expanses can act as underground dispersal high- ways for cave limited species (Barr, 1968). The purpose of the present study was to examine electrophoreti- cally several local populations of each of the other three subspecies of N. tellkampfi. We were interested in determining how infrasub- specific variation in these subspecies compared with that of nomi- nate tellkampfi. Further, we wished to use these electrophoretic data to quantitatively assess relationships among subspecies and also to gain some insight to how the present distributional pattern of the species has been produced. In these regards, Barr's (1979b) morpho- logical and biogeographic work provides a model against which the electrophoretic data can be examined.




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19861 Kane & Brunner-Neaphaenops tellkampfi Electrophoretic data gathered from a total of 18 populations (Fig. 1) of Neaphaenops tellkampfi were analyzed in this study. All of the electrophoretic data for ten of these populations were gathered dur- ing the course of the present study, between 1980 and 1983. These ten populations include three each of N. t. henroti (BL, CW and T/SS; Fig. l), N. t. meridionalis (H, OS and ST; Fig. 1) and N. t. viator (C, CB and S; Fig. 1) as recognized by Barr (1979b). The tenth population (F; Fig. 1) is a purported meridionalis X tellkarnpfi hybrid on morphological grounds (Barr, 19790). Most, but not all, of the electrophoretic data on the eight populations of N. t. tell- kampfi (B, BH, GO, HA, LB, P, RB and WH; Fig. 1) were col- lected in 1977-78 and reported by Turanchik and Kane (1979). Modifications of and additions to the nominate tellkampfi data set will be discussed in appropriate sections below. All 18 of the popula- tions sampled, with the exception of the SS and T sites of henroti, represent a single cave location. During the course of the study permission to sample the SS site was rescinded before a sample adequate for complete electrophoretic survey could be obtained. Subsequently the nearby T site was located but it harbored a much smaller henroti population and failed to yield a large enough sample to obtain data on all electrophoretic loci. Pooling of the data from the two sites, which appears to be justified by their geographic proximity, did produce a complete set of electrophoretic data. Beetles were maintained alive at 5OC or frozen at -80å C prior to electrophoresis. All electrophoresis was conducted on vertical polyacrylamide slab gels using an Ortec Model 4200 Electrophoresis System or a Hoefer Scientific SE600 System. Sample preparation and run procedures used in this study were similar to those dis- cussed by Giuseffi et al. (1978) and Turanchik and Kane (1979). Each animal provided enough homogenate for two assays. Six enzyme systems provided a total of seven consistently scora- ble loci. These included: alkaline phosphatase (ALP) (1); esterase (EST) (1); malate dehydrogenase (MDH) (2); phosphoglucomutase (PGM) (1); phosphoglucose isomerase (PGI) (1); and, xanthine dehydrogenase (XDH) (1). In addition a general protein (GP) stain revealed two sets of consistently scorable bands which are also



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Psyche [vo~. 93
included in the data. The more complete data of this study suggested interpretational changes at two loci from those reported by Turan- chik and Kane (1979). The present data show that the ALP bands are properly interpreted as a single variable locus rather than as two separate loci. Also, we have chosen a more conservative interpreta- tion of the XDH data. Electrophoretic analysis of XDH in N. tell- kampfi populations produces a single band per beetle with slight differences in mobility between some individuals. Initially these data appeared to be consistent with data reported by Singh et al. (1976) for a variable XDH locus in Drosophila pseudoobscura. However, application of additional techniques which Singh et al. (1976) used to reveal multiple bands in D. pseudoobscura heterozygotes, failed to reveal any multiple banded N. tellkampfi individuals at the XDH locus. More recently, Finnerty and Johnson (1979) have shown that data such as these may be the result of post-translational modifica- tion of an enzyme encoded by a monomorphic locus. We have chosen this interpretation of the XDH locus in the present study. PGM was not assayed in previous studies of N. tellkampfi (Giuseffi et al., 1978; Turanchik and Kane, 1979) and therefore populations of N. t. tellkampfi were re-collected and surveyed for this enzyme. The majority of the data analysis was accomplished using a Fortran 77 version of the BIOSYS-1 program developed by Swofford and Selander (198 1).
Of the nine putative genetic loci examined in this study, five were polymorphic and the remaining four were monomorphic with the same variant fixed in all populations of the four taxa (Table 1). Genetic variability in N. tellkampfi populations has been estimated as the proportion of polymorphic loci per population (P) and the average frequency of heterozygous loci per individual (H) (Table 2). The average N. tellkampfi population is polymorphic at approxi- mately 30% of its loci and the average individual in such a popula- tion is heterozygous at 9.4% of its loci (Table 2). These values are somewhat lower than those reported previously by Turanchik and Kane (1979) as a result of the addition of another invariant locus (PGM) and the more conservative interpretation of the XDH locus. However, these values of P and H still approach values typically reported for many surface invertebrates (Selander, 1976). Therefore,



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-
a 00
Table 1.
Gene frequencies for 18 populations of Neaphaenops tellkampfi. N = Sample size. 0
u
Cave
Locus CW BL T/SSH OS ST F B BH GO HA LB P RB WH C CB S 1. Monomorphic loci with the same variant fixed in all populations-4 loci (GP-2; MDH-1; PGM; XDH) 2. Loci coding for monomorphic and/ or polymorphic proteins. ALP N27 29 10 25 42 11 7 15 20 14 10 11 13 16 10 21 9 22 fio a 0.41 0.43 0.45 0.48 0.39 0.55 0.57 0.67 0.78 0.54 0.30 0.46 0.58 0.59 0.70 0.52 0.33 0.61 & b 0.59 0.57 0.55 0.52 0.61 0.45 0.43 0.33 0.22 0.46 0.70 0.54 0.42 0.41 0.30 0.48 0.67 0.39 5 s
EST N20 27 11 30 25 12 8 21 22 40 31 29 33 21 15 21 9 22 2 a 0.00 0.00 0.00 1.00 1.00 1.00 0.75 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1 c^
MDH-2 N 33 36 10 59 33 11 18 23 28 26 20 24 29 37 20 22 18 30 -+ a 0.91 0.93 1.00 0.89 0.95 1.00 1.00 0.94 0.79 0.83 0.90 0.96 0.91 0.77 0.93 0.93 1.00 0.97 $
b 0.09 0.07 0.00 0.11 0.05 0.00 0.00 0.06 0.21 0.17 0.10 0.04 0.09 0.23 0.07 0.07 0.00 0.03 Q 3
PGI N38 43 9 29 30 13 18 20 22 42 30 24 20 24 22 30 23 27 5 a 0.00 0.00 0.00 1.00 1.00
1.00 0.06 0.00 0.00 0.00 0.00 0.00
0.00 0.00
0.00
0.00
0.00 0.00
b 0.00 0.00
0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 1.00 1.00 1.00
c 1.00
1.00 1.00 0.00 0.00 0.00 0.94 1.00
1.00 1.00 1.00 1.00 1.00 1.00
1.00 0.00 0.00 0.00




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238 Psyche [VOI. 93
these data continue to support the contention that cave isolation does not necessarily result in a permanent reduction in genetic vari- ability for a species (Barr, 1968; Giuseffi et al., 1978; Dickson et al., 1979; Turanchik and Kane, 1979).
Estimates of P and H by subspecies (Table 2) indicate no differ- ences in genetic variability between the four taxa. Of the five polymor- phic loci examined, two, EST and PGI, are diagnostic of subspecific differentiation (Table 1). Three variants have been detected at each of these loci, with all meridionalis populations fixed for a slow migrating electromorph at both loci, all viator populations fixed for electromorphs of intermediate mobility at each locus, and all hen- roti and nominate tellkampfi populations being fixed for the fast migrating electromorphs at both loci. The only local population that is polymorphic at these two loci is population F (Fig. 1 and Table 1). This population, which is morphologically intermediate between meridionalis and tellkampfi and a purported hybrid of the two subspecies (Barr, 1979b), contains both the slow and fast elec- tromorphs at both the EST and PGI loci. The fact that these elec- tromorphs are alternatively fixed in meridionalis and tellkampfi populations respectively provides biochemical evidence of the hybrid nature of this population. By contrast, all three of the viator populations are fixed for the intermediate mobility electromorphs at both the EST and PGI loci even though two of these populations, C and S, lie in the zone of morphological intergradation between tell- kampfi and viator (Barr, 1979).
Rogers' (1972) estimate of genetic similarity (S) was used for pairwise comparisons of the 18 populations (Table 3). Rogers' dis- tance values were used in a UPGMA clustering procedure to pro- duce a biochemical dendrogram (Fig. 2). Infrasubspecific genetic identities are all greater than 0.90. This includes some populations, such as the C population of viator, separated from other popula- tions of the same subspecies by shallow rivers such as the Green River. This finding is consistent with earlier work (Turanchik and Kane, 1979) on populations BH, RB and B of nominate tellkampfi and with findings on at least one other cave limited species in the same area (Laing et al., 1976), and serves to reconfirm the fact that rivers per se are not necessarily dispersal barriers for cave limited forms. Genetic differentiation between subspecies is substantial in some cases (Fig. 2). Neaphaenops t. meridionalis and N. t. viator



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Kane & Brunner- Neaphaenops tellkampfi
Table 2.
Genetic variability in four subspecies of Neaphaenops tellkampfU. P = average proportion of polymorphic loci per population; H = average proportion of heterozygous loci per individual.
H Avg. Alleles /
Subspecies Site P OBS. EXP Locus
henroti
AVG 0.296 0.099 0.094 1.171
meridionalis H 0.333 0.133 0.124 1.216
0s 0.333 0.1 19 0.1 13 1.20 1
ST 0.111 0.040 0.058 1.109
AVG 0.259 0.097 0.098 1.175
tellkampfi
AVG
viator
AVG
mer. X tell.
hybrid
Neaphaenops tellkampfi
AVG 0.302 0.094 0.097 1.162
OVERALL 0.556
show levels of similarity to each other and to the other two subspe- cies in the range of 0.69-0.78 (Fig. 2). Genetic similarity between henroti and nominate tellkampfi (S > 0.96; Table 3) is as great as similarity values among local populations within a subspecies - Although these two subspecies are the most similar of the four



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240 Psyche [vo~. 93
Table 3.
Rogers' (1972) coefficients of genetic similarity (S) for comparisons of four subspecies of Neaphaenops tellkampfi. Values shown are averages of pairwise comparisons of appropriate populations. Values in parentheses are the ranges of similarity values appropriate to each comparison. Subspecies
No. of
Subspecies Pops. N. t. h. N. t. m. N. t. t. N. t. v. henroti 3 0.975
(0.971 -0.978)
meridionalis 3 0.748 0.956
(0.732-0.766) (0.942-0.983)
tellkampfi 8 0.963 0.730 0.963
(0.928-0.982) (0.689-0.763) (0.9 1 7-0.995) mer. X tell. 1 0.878
0.833
0.865 0.770
hybrid (0.863-0.886)
(0.828-0.836)
(0.844-0.883) (0.750-0.781)
morphologically, this large a genetic similarity is somewhat unex- pected given the presence of the Hart Co. Ridge, an apparent geo- logical barrier between these two subspecies. Genetic differentiation within and between subspecies was exam- ined using F-statistics (Wright, 1978) and a Chi-square contingency analysis of heterogeneity (Workman and Niswander, 1970). Allo- zyme phenotype frequencies for the 18 populations were used to calculate genetic differentiation (i.e., F-statistics) in a hierarchichal manner (Wright, 1978). The two hierarchical levels are subspecies within species and local populations within subspecies. Since the hybrid F population could not be unequivocally assigned to either tellkampfi or meridionalis, it was considered as a fifth "subspecies" at that level of the hierarchy. Three loci (ALP; GPT-1; MDH-2) are variable in some or all local populations of each subspecies. Signifi- cant heterogeneity in gene frequencies (Chi-square) was observed among N. t. tellkampfi populations at the ALP and MDH-2 loci but not at the GPT-1 locus (Table 4). Significant heterogeneity in gene frequencies at the GPT-1 locus was observed among local popula- tions of viator and among local populations of meridionalis, but no differentiation was observed among local populations of either sub- species at the ALP or MDH-2 loci (Table 4). No heterogeneity in gene frequency was observed among henroti populations at any of



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Kane & Brunner- Neaphaenops tellkampfi
ROGERS' Dl STANCE
Figure 2.
UPGMA dendrogram of 18 populations of Neaphaenops tellkampfi, generated from Rogers' genetic distance values for nine biochemical loci. the three variable loci. The slightly greater differentiation observed among tellkampfi populations may be due to the fact that this sub- species has a somewhat larger geographic range than any of the other three subspecies, or simply to the fact that more populations (8) were examined for nominate tellkampfi than for any of the other three subspecies.
Whereas genetic differentiation between infrasubspecific popula- tions is slight to moderate, differentiation between subspecies is very great (Table 5). At the level of subspecies, variation is observed at the EST and PGI loci in addition to the three loci discussed above. Significant heterogeneity in allele frequency between subspecies was observed at all five loci (Table 5) and overall genetic differentiation is very great (Fst = 0.528), with the EST and PGI loci essentially fixed for alternative alleles in three of the four subspecies.



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242 Psyche [vo~. 93
Table 4. F-statistics and heterogeneity chi-square values for four subspecies of N. tellkam p fi.
SUBSPECIES
henroti
meridionalis
tellkampfi
viator
henroti
meridonalis
tellkampfi
viator
henroti
meridionalis
tellkampfi
viator
ALP LOCUS
GPT-1 LOCUS
MDH-2 LOCUS
ns = P > 0.05; * = P < 0.05; *** = P > 0.005 FIT = correlation between uniting gametes relative to the gametes of the total population
Fig = average correlation over subdivisions of uniting gametes relative to those of their own subdivision
FsT = correlation of random gametes within subdivisions relative to gametes of the total population
Slatkin (1981) has proposed a method to estimate overall gene flow in natural populations in a qualitative manner from gene fre- quency data. Using computer simulation, Slatkin (198 1) has demon- strated a dependence between gene flow and the conditional average frequency of an allele, p(i) where:
d = number of demes sampled
i = number of demes in which the allele occurs p = average frequency of the alleles in those demes Caccone (1985) used Slatkin's technique to assess gene flow in several species of cave animals, based on her own data for H. sub- terraneus, the data of Laing et al. (1976) for the scavenger beetle Ptomaphagus hirtus and Turanchik and Kane's (1 979) data for the