Aust. J. BioI Sci., 1987, 40, 417-33

Electrophoretic Resolution of Species Boundaries in Australian Microchiroptera. 111*. The Nycticeiini - Scotorepens and Scoteanax (Chiroptera : )

P. R. Baverstock, M. Adams, T. Reardon and C. H. S. Watts Evolutionary Biology Unit, South Australian Museum, North Terrace, Adelaide, S.A. 5000.

Abstract Two hundred and sixty two specimens of of the tribe Nycticeiini were sampled from throughout Aust­ ralia, and their tissues subjected to aliozyme electrophoresis with a view towards delineating species bound­ aries. A total of 30 loci were resolved, detailed analysis of which revealed five species - greyii, sanborni, balstoni, orion and rueppellii. The specific boundaries recognized differ from ali previous treatments of Australian members, but are supported by the taxonomic arrangement proposed by Kitchener and Caputi (1985). The data also support separate generic recognition of rueppe/lii. Finally, the genetic data reveal a high level of population sub structuring in these bats.

Introduction The species-level of the Australian Microchiroptera has been in a state of con­ siderable confusion and flux for the last 100 years. This has been especially true of the Aust­ ralian Nycticeiini. This problem was recognized by the Australian Biological Resources Study (ABRS) who, in 1980, placed as one of their preferred objectives the species-level taxonomy 'of Australian Microchiroptera (especially ),. The Evolutionary Biology Unit has applied the technique of allozyme electrophoresis to species-level taxonomy of Australian Microchiroptera. The present paper on the Australian Nycticeiini is part III in the series of these studies. Parts I and II are Adams et al. (1987a, 1987b). Named forms of Nycticeiini in Australia include greyii Gray, 1843; Nycticeius rueppellii Peters, 1866; Scoteinus balstoni Thomas, 1906; Scoteinus influatus Thomas, 1924; Scoteinus orion Troughton, 1937; Scoteinus sanborni Troughton, 1937; Scoteinus balstoni caprenus Troughton 1937; and Scoteinus orion aquilo Troughton, 1937. Numerous specific and generic arrangements of these named forms have been used in the literature (see Kitchener and Caputi 1985). Ride (1970) recognized that this group, along with many other groups of Australian , had been 'oversplit' and consolidated it into only three species - N. rueppellii, a morphologically distinctive large species; the small N. greyii from throughout most of main­ land Australia; and N. influatus, similar to N. greyii but larger and then known from only the type specimen from central Queensland. Koopman (1978) split Ride's (1970) greyii into two species and six subspecies - N. greyii, N. balstoni balstoni, N.b. caprenus, N.b. sanborni, N.b. orion and N.b. aquilo, a view held largely unchanged by Koopman (1984). Hall and Richards (1979) generally followed Koopman's arrangement for eastern Australia, and Aitken (1981) added distributional records using Koop­ man's scheme as a framework. Our interpretation of Koopman's framework is represented by the species' distributional patterns shown in Fig. 1. This pattern formed the null hypothesis

*Part II, Aust. 1. BioI. Sci., 1987, 40, 163-70. 0004-9417/87/040417$02.00 418 P. R. Baverstock et al.

against which we tested the electrophoretic data (see Adams et al. 1987a for the rationale of this approach). The present study is based on an analysis of 30 loci in 262 specimens of Aust­ ralian Nycticeiini.

subspecies of N. balstoni N. greyii

N. rueppellii N. balstoni N. influatus

Fig. 1. Our diagrammatic interpretation of species boundaries in Australian Nycticeiini based on Koop­ man (1978) and Hall and Richards (1979). This pattern formed the null hypothesis for this study.

Based upon morphometric analyses in conjunction with the present electrophoretic analyses, Kitchener and Caputi (1985) presented a formal taxonomic revision of the group in Australia. We follow their nomenclature here. They recognize five species in two genera, Scoteanax rueppellii and Scotorepens orion, balstoni, sanborni and greyii.

Materials and Methods Specimens were collected from as broad an area of Australia as practicable, with more emphasis being placed on geographic spread of collecting than on numbers of specimens (Adams et al. 1987a). Specimens were collected in two bouts of sampling. The first round was made in 1981, and the tissues subjected to preliminary electrophoretic analysis to provide a rough guide as to the number of genetic groups. Based on these results, a second round of collection was made in 1982. Additional specimens were donated by other researchers, to extend the geographic area sampled. Details of tissue sampling tech­ niques and electrophoresis follow Adams et at. (l987a). Electrophoretic Resolution of Species Boundaries in Microchiroptera. III. 419

Because sampling was concentrated over such a broad geographic range, 94 individual localities were represented. For the convenience of analysis and presentation, all of the specimens from a single locality were treated as a 'local sample', local samples from the sample phytogeographic region (following Kitchener and Caputi 1985) as a 'subpopulation': and subpopulations from the same general area and of the same genetic type (i.e. shared alleles at all loci) as a 'population'. These populations formed the 19 Operational Taxonomic Units (OTUs) as the basis for analysis. Full collecting details are given in an Accessory Publication* and summarized in Figs 2-4.

!------,

o, , 500, km Fig. 2. Localities from which Scotorepens sanborni was sampled showing phytogeographic regions (num­ bers) and population codes (letters).

Results A total of 30 loci yielded staining of sufficient intensity and resolution to be reliably inter­ preted. The proteins used were: aconitate hydratase (ACON, EC 4.2.1.3), adenosine deaminase (ADA, EC 3.5.4.4) alcohol dehydrogenase (ADH, EC 1.1.1.1), adenylate kinase (AK, EC 2.7.4.3), carbonate dehydratase (CA, EC 4.2.1.1), enolase (ENOL, EC 4.2.1.11), fumarate hydratase (FUM, EC 4.2.1.2), aspartate aminotransferase (GOT, EC 2.6.1.1), glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), glycerol-3-phosphate deyhydrogenase (GPD, EC 1.1.1.8), glucosephosphate isomerase (GPI, EC 5.3.1.9), guanylate kinase (GUK, EC 2.7.4.8), isocitrate dehydrogenase (IDH, EC 1.1.1.42), lactate dehydrogenase (LDH, EC 1.1.1.27), malate dehydrogenase (MDH, EC 1.1.1.37), mannosephosphate isomerase (MPI, EC 5.3.1.8), purine nucleoside phosphorylase (NP, EC 2.4.2.1), peptidases (PEP, EC 3.4.11.* or 3.4.13.*), 6-phosphogluconate dehydrogenase (6PGD, EC 1.1.1.44), phosphoglycerate kinase (PGK, EC 2.7.2.3), phosphoglucomutase (PGM, EC 2.7.5.1), pyruvate kinase (PK, EC 2.7.1.40), L-iditol dehydrogenase (SORDH, EC 1.1.1.14) and triosephosphate isomerase (TPI, EC 5.3.1.1). Thble 1 shows the allelic profiles of the 19 populations for the 30 loci. These data were then converted into a matrix expressing the percentage of loci showing fixed differences between populations (Adams et at. 1987a) and a matrix of Nei D values corrected for small sample sizes (Nei 1978) - Table 2. Phenograms constructed by Average Linkage Cluster (ALC - Sneath and Sokal 1973) for both matrices are shown in Figs 5 and 6. Populations Rand S (rueppellil) stand well apart genetically from all other populations, differing at an average of 59070 of loci and an average Nei D of 1.05. By contrast, populations Rand S are genetically very similar for the 30 loci used. Over a distance of 2200 km, the populations show no fixed allelic differences and a Nei D of 0.00.

*Available from the Managing Editor, Australian Journal of Biological Sciences, 314 Albert St, East Melbourne 3002, Australia. 420 P. R. Baverstock et al.

J

o, km

_./--~Q

Fig. 3. Localities from which Scotorepens greyii (e), Scotorepens orion (_) and Scoteanax rueppellii (.6) were sampled showing phytogeographic regions (numbers) and population codes (letters). Populations A to Q are remarkably uniform genetically. The largest percentage fixed differ­ ence is 11070 of loci, and the corresponding Nei D values are around 0.25 (Table 2). These differences are not, by themselves, sufficient to indicate that populations A to Q belong to anything other than a single species (Baverstock et al. 1977; Thorpe 1983). However, a more detailed analysis of fixed differences in sympatry indicates the presence of several distinct biological species. In assessing the specific status of these populations, it is instructive to begin with a sample of eight bats (BI65-BI72) collected on one night, in the same mistnet at Berry Springs in phytogeo­ graphic region 18. In this sample, two genetic groups were evident, one group of five individuals and one of three individuals. The two groups exhibited fixed allelic differences at four loci - Acon-2, 00t-2, Mpi and Pep-B (Thble 3). If these eight individuals had been taken at random from a single population, each locus would be in Hardy-Weinberg equilibrium. The estimates of p and q are, therefore, p = 0.63 and q = 0.37 and the expected frequency of heterozygotes at anyone locus is 2pq = 0.47 (for Acon-2, alleles c and a can be treated as a single allele for these calculations). The probability of not drawing a heterozygote among eight individuals at anyone locus is then (1 - 0.47)8 = 0.006. The simultaneous probability of concordance at all four loci becomes (0.006)4 = 1.5 X 10-9• The eight individuals clearly do not belong to a single random breeding population. A reasonable alternative hypothesis is that the sample consists of two biological species, characterized by different allelic profiles for Acon-2, 00t-2, Mpi and Pep-B. Indeed, when the eight individuals are separated on this basis, numerous other allelic differences become apparent (Table 3). For example, for Ada, Adh and 00t-1 the two forms carry different major alleles, while for Fum, Opi and Pk, alleles at a relatively high frequency in one group are absent from the other. Electrophoretic Resolution of Species Boundaries in Microchiroptera. III. 421

c:;,. ~t· • 30 •• 23 31 31•

o, , 500I km

Fig. 4. Localities from which Scotorepens balstoni was sampled showing phytogeographic regions (num­ bers) and population codes (letters).

These eight individuals are members of populations B (consisting of 6 individuals) and H (consisting of 24 individuals). When all 30 individuals are considered (Table 1), occasional heterozygotes are found at either Acon-2 or at Pep-B, two of the four 'diagnostic' loci. However, all 30 individuals are assignable to one or the other groups on at least three diagnostic loci. On the phenograms (Figs 5 and 6), population B clusters with populations A, C, and D while population H clusters with populations E, F, G, I, J, K, and Q. It remains to be seen how these 'diagnostic' loci hold up across the geographic range. In the north-west, populations A (n = 17) and F (n = 24) are broadly sympatric. Here Mpi is again a fixed difference, as is Acon-2. A single heterozygote was found for 00t-2, but this individual was clearly assignable on Mpi and Acon-2. When the 41 individuals are separated on this basis, numerous other large allele frequency differences are apparent at other loci such as Adh, Ca, Oot-l, Np, and Pep-B. On the east coast, populations D and J occur within 180 km of each other. Here again, both Mpi and Acon-2 exhibit fixed differences. Numerous other loci show large differences in allele frequency, e.g. Adh, Ca, Fum, Oot-l, 00t-2, Ouk, Np, and Pk. Of the remaining populations, none is strictly sympatric. However, population C clearly lies with populations A, B, and D on the basis of its Mpi. Numerous other loci show strong concordance, such as Acon-2, Adh, Fum and 00t-2. Similarly populations E, G, I, and K clearly align with populations F, H, and J. Thus for populations A to K, a rather clear picture emerges of two species. One (consisting of populations A to D) occurs on the east coast from Rockhampton, Cape York, and the coastal strip from Darwin, N.T. to Cape Bossut, W.A. The other (populations E to K) occurs Table 1. Allele frequencies, expressed as a percentage, for the 19 populations at 30 loci ~ Alleles are designated alphabetically (a is the ijlost cathodically migrating). Where an enzyme is encoded by more than one locus, loci are designated N numerically according to increasing electrophoretic mobility. 2N (in parentheses) is the number of haploid genomes sampled. The following loci were invariant: Ak-2, Mdh-l, Mdh-2, Pgk.

sanborni qreyii balstoni orion rueDDellii

A B C D E F 6 H I J \( L 1\ N 0 P Q R S LOCUS 1341 (12) Ib2) Ibl (41 (4B) 1221 14BI (521 1561 (14) (12) 13BI (14) 1301 (56) Ilbl 141 (4)

Acon-l b b b(84) b b b b b b(97) b b b b b b b b c c aHb) a (3)

Acon-2 UbI e i(3) e d d d(47) f (2) d(29) f 191 d171 i (12) f I1b) e 13b) i(7) f <1B)i(7) i h(1S) g(21 c(4S1 el41 c(b91 d(201 c(93) e(50) e16B) d(b41 e(43) e(51) elBbl eml e19S) b(4) d(10) a(2) c(1) dl3S1 d(6) dlS01 d(311d(7) al41 cUbl bIb) am

Ada c(BB)C(921 c c d(50) dl101 d(73) d(711 dllb) dl41 c c C t t C t (94) a a b 1121b (B) elS0) c (90) c (27) c (29) elS2) c 19b) blbl bl21

Adh f 1941f i (21 f f(2S) fIb) f(27) fIB) i(21 i(b) f(14) f(33) h(3) f(22) f(17) f(2) g(94) e h(2S1 :-c

d Ibl f 18ll e1S0) e1741eml e(S4) fIlS) f(7) elSb) e(b71 fIlS) e17S1e(S3) eIBS) fIb) e17S) ?C b (251 (4) (2) 1771 (b9) (791 (131 b::I el101 c c e e e d I>l < dIS) bUb) bIb) c(2) c(1b) d(3) ....., ~ c(2) b(2) b(2) 0 (') a(2) ~ .,~ :- t!1 Ak-l b b b b b b b b b b b b b a a C1I b b b b ..,~ 0 ::r Ca d(24) e(B) e(SB) !!(S0) f(2S) f(24) f(S4) f(19) f(S4) f(S1) f(72) f(B3) f(B1l flBb) 9(4) f (bB) d h h '"0.., a (7b) di92) d 142i d(S0) d (7S) d (7b) d(411 d (71) d(42) d (43) d (14) d (17) d(19) d(14) f (9b) d (321 ~ "'. a(S) c (21 a(4) e(2) e(7) C1I bIb) b(41 b(7) '"0 a(2) §. 0 ::;

0...., Enol C(3) b(92) d(21 b b b b(9bl b b (9S) b (9b1 b b b b b b b b b Vl b (97) a (B) b(9S) a(41 a(21 a(41 '"o.C1I ~ t:l:l (30) (231 (S01 e(73) (3S) (b0) 0 FUI e(291 e(S8) e(90) e b e(21 C(5) e(2) c(4) e(13) e(B) e e c c e c c I'! ::; b(71) bIB) b 1101 b(921 b(9S) b(9S) b(94) b(B71 b(92) b(70) b(77) b(501 b(271 b(60) bl401 P- I".., (42) alb) a(2) a(2) n· '"s· 6ot-l d (97) e (2S) f (3) f(33) d e(6) e(SS) e(B4) e(79) e(65) e(571 e(B2) f (3) e (931 e(90) f (31 d d d ~ "'..., a(3) d(67) e(87) e(67) d(92) d(45) d (121 d(21l d (3S) d(43) d(9) e(84) c(7) d(6) e(B2) 0 d(3) n cIS) dIS) c(2) c (2) c(91 cl41 d<111 ....e: 0 b(21 b(2) c(4) '"

b(21 66pd a a b 121 a a a a a a a a a a a a a b b a(9BI al981

§a1 b b b b b c(2) b cl21 b d(4) b b b b b c(5) b a a .j>. b(981 b(98) b(96) b(951 N w .j:>. §.Qi d(3) d (33) d (2) c(3) d(b) tv b b b b b b b b a b dUll b c c .j:>. b (97) b (b7) b (98) a(97) b(94) b(87) a(2)

Guk b b b b b b b b b 0(64) b b b b b b b b b a(36)

Idh d(3) c ( c e f (2) c e(2) c c f(7) c ( c ( f (5) f (B) b b c (97) e(2) c (98) C(87) (95) C(92) c(92) albl a(4)

Ldh-l a a a a a a a a a a a a a a a a a b b

Ldh-2 c c ( C I: (50) (9B) I: d(2) c(9B) (9B) c(7B) c I: ( C C I: C c b (50) a(2) 1:(94) b(2) b(2) b(22) b (4)

!1U e e e(98) e I: d(4U d(5) c c c ( f (931 f g(25) h h b(2) (59) (95) e(7) b(75)

Hi I: ( d(34) d(B3) I: d(52) d (9) d(23) eIB) e(14) d(42) c I: (B2) I: ( c (B4) (43) I: I: I:lb6) c1(7) c(24) 1:(77) c(711 d1(4) d(41) (lSI) alIB) allb) a(S7) :-0 a(24) a(14) a(6) (76) (45) b(71 ?" I:!:l b(2) ~ <: !:l f!< 0 Pep-B d(3) a d (90) d d e(2) d e(4) d e(2) d d d d d (96) d d(94) ( ( n ~ a(97) (9B) b(4) a!101 d d 1921 dl891 alb) ~ a(4) I: (2) :-'" a (7) trI (b (80) (14) (70) (45) (50) bPgd f f IB0) f f(94) f gl2t f(94) f(94) f f f(94) h f f f 9 9 ~ 0 c(20) d(3) f (50) d(4) d(4) c(50) '0 c(20) c(b) flb9) e(21 c(b) ::r 0 d(4) c(4) c(3) c 13b) c (23) ((4B) .... g, c (19) b(3) b (3) n b(4) ::>:l '"0> a(2) 0 ao· ::s Pgm-l (94) d(8) dIS) ( c d(4) d(5) d(4) d(bl c c c d(b) d(7) d(4) c c d d 0..., Ul bib) c (92) e19S) c(92) c19S) el9b) c(92) c (911 e (93) t(92) '0 a(4) a(2) a(3) a(4) ;;;.'"n 0> t:I:I 0 d19S) d(93) d(90) d(93) e(20) - s:: Pgll-3 d(97) d e(2) d d17S) d(9S) dl91l d d(98) d d d a ::s P- d(98) b (2S) b (S) e(9) t(2) b(S) b(]) b(10) tiS) dm) I>' c(3) ::l. a(2) b (10) rJl 5· a:: d(3) d(3) ( d(32) d(44) eUll e(4S) e(SB) c(/i1) d(50) d(79) d(49) d(52) t(94) a a n· Pk c e c .... 0 c(97) c(97) c(68) c(Sb) d(17) d(29! d(281 b(391e1S0! e(21) c(47) c(44) bIb) n ::r::;. c(72) c(2b) (14) b(4) b(4) 0 -g (1) .... c(12) b(91) (Ib) b (95) b b b b e ( !" Sordh b b b b b (6) b b b ...... b (88) a (9) b(94) b(94) a(S) ....

I2i ( c c ( c c c c ( c(94) ( a a a a a c c c bIb)

The following loci were invariant: Ak-2, "dh-l, "dh-2, and .~.

t; v. .j:>. N 0-

Table 2. Matrix of genetic differences among the 19 populations of Nycticeiini The upper matrix is the percentage of fixed differences. The lower matrix shows the corrected Nei D values (x 102)

sanborni 9!.fiU.. balstoni ori ruep

A B C D E F 6 H I J K L II N 0 P Ii R S A 6 6 .) 7 7 7 3 7 7 7 16 13 13 13 Ie 3 59 613 "/ B 4 0 3 13 13 13 7 13 16 13 13 17 13 17 13 'J 59 60

C 16 7 13 7 7 7 3 7 7 7 16 13 Ie 13 Ie 13 4B 513 D 13 8 1 13 7 7 3 7 7 7 16 13 16 13 167 66 67

E 19 23 213 23 6 13 13 13 13 13 13 13 13 7 Ie 3 62 63

F 21 24 18 IB 3 13 0 6 6 13 13 13 Ie 7 Ie 3 52 53

G 21 25 17 26 3 5 13 0 13 6 13 13 Ie 7 Ie 3 55 57 H 25 27 18 21 7 9 1 6 6 6 13 13 16 7 16 3 48 50 I 21 24 15 IB 5 6 1 2 6 6 13 13 10 7 10 3 52 53

J 23 26 17 19 B 7 4 4 2 13 13 13 Ie 7 10 3 55 57 K 25 29 19 21 9 9 4 5 2 1 13 13 Ie 7 10 3 59 613 ., L 27 27 21 24 23 2320 23 IB 21 21 0 .J 3 0 16 59 60 II 30 30 ..'H,' 26 26 24 21 23 19 21 21 1 3 3 0 13 55 57 N 36 30 20 24 23 22IB 20 17 18 19 5 5 6 0 13 62 63 0 28 27 18 22 23 22 18 213 16 18 195 5 6 0 13 55 57 p 2B 28 19 23 23 21 18 18 16 18 18 5 4 6 1 10 59 57 :-c Ii IB 16 13 15 i3 11 16 18 1617 19 27 2B 26 25 22 59 66 ?O R 1013 98 1132 112 162 106 99 1132 101 1137 164 1139 111 leB 1134 1137106 13 tl:I po < S 103 lee 1135 114 1135 1135 1133 107 1136 111 109 113 115 112 108 112 1132 0 (\) ;;l ()0 i'<"

~

1'?- Electrophoretic Resolution of Species Boundaries in Microchiroptera. III. 427

in the more arid areas of northern and eastern Australia, excluding Cape York but including the north and north-west coast. Only one locus is totally diagnostic for these species - Mpi. Other loci that are diagnostic in some areas (Acon-2, Pep-B and Got-2) occur as sporadic heterozygotes in other areas. There are two possible explanations for this situation. One possibility is that there has been introgression between the two species due to hybridization. If this is the explanation, then the introgression is limited since Mpi remains diagnostic and the remaining three loci are well out of Hardy-Weinberg equilibrium taken over both species. Alternatively, Acon-2, Pep-B and Got-2 were polymorphic in the common ancestral population, and have subsequently gone almost to fixation in the two species. Under either model, it is still apparent that two biological species are involved. Fixed differences (%)

0 10 20 30 40 50 60 70 I I I i i i I i

A

B sanborni C t-- O.

E

F r---- G

greyii H ~

J t-

K orion .a-

balstoni p. f------' :} RTr----_____------' rueppellii I S. Fig. 5. Phenogram constructed by Average Linkage Cluster showing percentage fixed differences between Operational Taxonomic Units.

Population Q (east of the Great Dividing Range in New South Wales and Victoria) is allopatric with populations A to K. On the phenogram of fixed differences, it is very similar to the E to K group, although on Nei D values it stands further apart. The following detailed locus by locus analysis reveals that it is quite distinct from its geographically nearest population - population K. There is a fixed difference at Mpi, with population Q carrying alleles which 428 P. R. Baverstock et al.

were found in no other population (a single individual in population C was heterozygous (for allele b). A number of other loci show very different allelic patterns for populations K and Q, e.g. Acon-2, Adh, Ca, Fum, Got-i, Np, 6Pgd, and Pk. The net effect is that populations K and Q have a Nei D of 0.191 (Table 2). The phenogram is a little deceptive here in showing the average Nei D between population Q and the E-K group as 0.15. The reason for the dis­ crepancy is that many of the alleles carried by Q that differentiate it from K are shared with population E in the west (e.g. see Ca, Got-i and Pk). Corrected Nei 0

o ()'2 04 08 1·2

sanborni I::

G

greyii H

J

K

orion • a_--...J

L

M

balstoni P

N o

rueppellii I Rs f------.....J Fig. 6. Phenogram constructed by Average Linkage Cluster showing corrected Nei D values between Operational Taxonomic Units.

The final populations to consider are L, M, N, 0 and P. As a group these populations are genetically very similar. They share alleles at all loci except one - Gpi. Here, populations Land M carry alleles a and c, whereas populations N, 0 and P carry alleles band d. (A single individual from population P was heterozygous for the a allele.) The LMNOP group is specifically distinct from both the ABCD group and the EFGHIJK group. Thus populations Nand 0 overlap with population C in southern Cape York. Here, there are fixed differences at Got-2, Mpi, and Tpi. The absence of heterozygotes for three ._-----...,------'

Electrophoretic Resolution of Species Boundaries in Microchiroptera. III. 429

loci is conclusive evidence of their specific distinctiveness. Similarly, the group of populations MNOP overlap the populations 11K. Here fixed differences are found at 00t-2 and Tpi, while for Mpi, only population 0 shares an allele at a low frequency with 11K.

S. sanborni S. greyii

S. balstoni S. orion

Fig. 7. Species boundaries in Australian Nycticeiini as interpreted from this study (S. rueppellii as per Fig. 1). We did not find the LMNOP group anywhere sympatric with population Q. The two groups differ at an average of 12% of loci and an average Nei D of 0.26.

Discussion The present study utilizing allozyme electrophoresis of 30 loci has revealed five genetic groups, including a minimum of four species, among the 262 specimens for which frozen tissues were available. The populations corresponding to these four species are ABCD, EFGHI1K, LMNOP, and RS, a conclusion based upon the presence of at least two fixed genetic differences in sym­ patry between each pair of groups. Population Q stands apart from all four groups, but the specific status of population Q is not clearly resolved by the electrophoretic data, since it is sympatric only with RS (from which it is clearly distinct at the specific level), and allopatric with the remaining three groups. Kitchener and Caputi (1985) have conducted an extensive multivariate morphological analy­ sis that included many of the specimens here subjected to electrophoresis, along with the types of the named forms greyii Gray, 1843; balstoni Thomas, 1906; influatus Thomas, 1924; orion Troughton, 1937; sanborni Troughton, 1937; balstoni caprenus Troughton, 1937; orion aquilo 430 P. R. Baverstock et al.

Troughton, 1937. They concluded that our five groupings correspond to these named forms in the following way: ABCD - sanborni EFGHIJK - greyii, balstoni caprenus, orion aquilo Q - orion LMNOP - balstoni, influatus RS - rueppellii Based upon their morphometric analysis, orion (our population Q) was clearly distinct specifically from both greyii (our group of populations E to K) and balstoni (our group of populations LMNOP). A summary of the picture in Australian Nycticeiini, as deduced from a combination of the electrophoretic and morphometric data is shown in Fig. 7. It is impor­ tant to realize that this revision is not merely raising to the rank of species some of Koopman's (1978, 1984) subspecies of balstoni, but a completely different view of what constitutes these species. Thus Koopman's balstoni sanborni becomes sanborni, Koopman's western greyii becomes sanborni, Koopman's balstoni caprenus becomes greyii, Koopman's balstoni aquilo becomes greyii, and Koopman's influatus becomes balstoni. It is now apparent why such taxonomic confusion has occurred in the past. Firstly, the species greyii, balstoni, and sanborni are morphologically very similar (Kitchener and Caputi 1985). Secondly, there is considerable clinal variation within species, especially for size. Thus, for example, sanborni in northern Australia becomes smaller from east to west, while greyii becomes larger. The result is that sanborni is larger than greyii in Queensland, but smaller than greyii in the north-west of Western Australia; they are indistinguishable by size in the Northern Territory. Indeed, in the Northern Territory, even discriminant function analysis failed to correctly identify all specimens allocated to species by electrophoresis (Kitchener and Caputi 1985). Yet there can be no doubt that two species are present in the Northern Territory, since it is here that the two are most distinctive electrophoretically (see Results). Thus greyii and sanborni are truly sibling species. The status of influatus warrants special comment, since most authors have recognized it as specifically distinct, based primarily on its large size. We are unable of course to conduct electrophoresis on the type specimen for influatus. However, we did have a single specimen from Hughendon (45 km from the type locality of Prairie), and two specimens from 16 km south. of Mt Isa (4 km north of the collection locality of a specimen ascribed to influatus by Koopman 1978, 1984). These specimens were certainly large for balstoni, with forearm lengths and condylobasal lengths exceeding those of specimens ascribed to influatus by Koopman. Yet we were unable to discern any allozymic distinctiveness of these specimens from balstoni. Of course, it is always possible that influatus and balstoNi are specifically distinct, but share alleles at all 30 loci studied here. However, except in some birds, we know of no confirmed case of two biological species failing to show any fixed differences at 30 loci. Therefore, a more parsimonious explanation at this stage is that the large specimens from central Queens­ land are the terminus of a size cline within balstoni. Indeed, the multivariate analysis by Kitch­ ener and Caputi (1985) shows such a continuous cline in morphology. It may be significant that, within balstoni, populations Land M show a virtual fixed differ­ ence from populations Nand 0 at the Gpi locus (see Results). Because populations LM and NOP are allopatric, the significance of the difference is not clear. However populations M and N came to within 400 km of each other. Thus if they are members of the same species, there would be a steep cline in Gpi allele frequencies between M and N. Intensive sampling in the region of the Queensland/Northern Territory border would be necessary to determine whether the fixed difference is maintained in sympatry, or whether a steep cline occurs. The high incidence of morphological clines in greyii, balstoni and sanborni led Kitchener and Caputi (1985) to suggest that they are relatively sedentary forms with little gene flow between geographic groups. The electrophoretic data provide strong support for such a scenario, since there are large allele frequency differences between populations at numerous Electrophoretic Resolution of Species Boundaries in Microchiroptera. III. 431

loci. For example within greyii, allele d is fixed for Acon-2 in the west (E and F), but drops to frequencies of 20070 and lower in the east (J and K). The sample sizes are large (2n = 48 in F and 2n = 56 in J) so that these differences are highly significant statistically. This pattern is repeated for many loci, e.g. in sanborni (Ca, Fum, Got-I, Got-2, Np, Pep-B), in greyii (Acon-2, Ada, Ca, Got-I, Mpi, Np, 6Pgd, and Pk), and in balstoni (Gpi and 6Pgd). Such a level of sub structuring is perhaps unexpected for a , in which high vagility might be anticipated.

Table 3. Allelic profiles at 30 loci for a sample of eight individuals from Berry Springs on 18 September 1982, split using Got-2 as a taxonomic character sanborni, EBU B165, B167, B168, B169, B170. greyii, EBU B166, B171, BI72. The remaining 13 loci were invariant sanborni greyii sanborni arnu. Locus (2N) (l0) (61 Locus (2N) 110) (6)

e 100 d 40 b 60 100 b 60 100 c 83 a 17 b 17 a 100 83 d 67 c 90 33 e 100 b 10 c 100

f 100 17 d 16 e 67 t 100 67 c 16 a 17

17 d 100 e 10 a 100 d 90 83 f 100 83 d 17 b 90 100 a 10 d 10 c 90 100 c 50 b 50 100 d 50 c 100 50 e 30 100 d 60 c 17 c 10 b 100 83

c 100 b 100

The substructuring observed may be due to the presence of discrete subpopulations, or to isolation-by-distance processes (see Richardson et af. 1986). Much larger sample sizes than we had available would be needed to distinguish between these alternative hypotheses. On morphology, rueppellii stands well apart from the remaining species, leading Kitchener and Caputi (1985) to place it in a genus of its own, Scoteanax Troughton, 1943. Our 432 P. R. Baverstock et al.

electrophoretic data would support separate generic recognition of rueppellii. Genetically, rueppellii stands well apart from the remaining species, differing by an average Nei D of 1.07, which is well beyond the average Nei D typical of congeneriC species of mammals (Thorpe 1983). The remaining species they placed in a single genus Scotorepens Troughton, 1943, separate from Nyceticeius Rafinesque, 1819. The electrophoretic data would support congeneric rank­ ing of sanborni, greyii, orion and balstoni but, in the absence of electrophoretic data on Nycticeius, we are unable to comment on generic separation of Scotorepens from Nycticeius. The species members of the genus Scotorepens are remarkably similar at the electrophor­ etic level. Thus orion and greyii have fixed differences at an average of only 3070 of loci, and sanborni and greyii at an average of only 6%. Scotorepens balstoni differs from the remain­ ing members of the genus at an average of only 12% of loci (Fig. 5). This pattern contrasts markedly with the situation found in and in the PipistrelluslFalsistrellus complex, where the different congeneric species show much higher levels of genetic divergence, up to 60% in Eptesicus and 30% in the PipistrelluslFalsistrellus complex (Adams et al. 1987a, 1987b). In the case of Scotorepens, we would not have been able to distinguish the different species if only allopatric populations had been sampled. Indeed, using electrophoretic data alone we would not recognize orion as specifically distinct from greyii, since they are nowhere sym­ patric. Here, the specific distinctiveness of orion is based primarily on morphological data (Kitchener and Caputi 1985). The present study illustrates the value of electrophoretic analysis for the delineation of species boundaries, especially when conducted in parallel with morphological studies. It is unlikely, for example, that the situation in Northern Australia would have been elucidated without the electrophoretic data. Indeed, even with the electrophoretic data, discriminant func­ tion analysis of greyii and sanborni failed to clearly distinguish the two forms where they are sympatric in the north-east Western Australia-Northern Territory area (Kitchener and Caputi 1985).

Acknowledgments We are grateful to the many people who assisted in this study by contributing specimens, especially S. Churchill, S. Flavel, W. Hollsworth, D. Kitchener, N. McKenzie, H. Parnaby, I. Temby, and C. Tidemann. Special thanks must go to L. Hall for providing the initial sampling strategy in 1981. We are also grateful to the State fauna authorities for their assistance with collecting permits, to local wildlife rangers, and to various landowners. R. Andrews and S. Donnellan kindly read and criticized earlier drafts of the manuscript. 1. Riede prepared the diagrams. We thank P. Kidd for typing the manuscript. This study was partly funded by the Australian Biological Resources Study.

References

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Manuscript received 10 March 1987, accepted 1 July 1987