COBISS: 1.01
AGE ESTIMATES FOR SOME SUBTERRANEAN TAxA AND LINEAGES IN THE DINARIC KARST
OCENE STAROSTI ZA NEKATERE PODZEMELJSKE TAKSONE IN ŽIVALSKE LINIJE NA DINARSKEM KRASU
Peter TRONTELJ1, Špela GORIČKI1, Slavko POLAK2, Rudi VEROVNIK1, Valerija ZAKŠEK1 & Boris SKET1
Abstract                                 UDC 575.8:551.442(234.422.1)
591.542(234.422.1)
Peter Trontelj, Špela Gorički, Slavko Polak, Rudi Verovnik, Valerija Zakšek & Boris Sket: Age estimates for some subter-ranean taxa and lineages in the Dinaric Karst
Using a comparative phylogeographic approach and diferent independent molecular clocks we propose a timescale for the evolution of troglobionts in the Dinaric Karst that is relatively consistent over a wide taxonomic range. Keystone events seem to belong to two age classes. (1) Major splits within holodinaric taxa are from the mid-Miocene. Tey present the potential up-per limit for the age of cave invasions. (2) Regional diferentia-tion, including speciation, which can at least in part be associated with a subterranean phase, took place from early Pliocene to mid-Pleistocene. we suggest two to fve million years as the time when most of the analyzed lineages started invading the Dinaric Karst underground.
Key words: subterranean, molecular clock, molecular phylog-eny, phylogeography, Dinaric Karst.
Izvleček                                 UDK 575.8:551.442(234.422.1)
591.542(234.422.1)
Peter Trontelj, Špela Gorički, Slavko Polak, Rudi Verovnik, Valerija Zakšek & Boris Sket: Ocene starosti za nekatere podzemeljske taksone in živalske linije na Dinarskem krasu
Z uporabo primerjalnega flogeografskega pristopa in neodvisnih molekularnih ur smo predlagali časovni potek evolucije troglobiontov Dinarskega krasa, ki velja za sorazmerno veliko število taksonov. Zdi se, da ključni dogodki pripadajo dvema obdobjema. (1) Glavne razdelitve znotraj holodinarskih tak-sonov so iz obdobje srednjega miocena. Predstavljajo zgornji potencialni časovni limit za naselitev jam. (2) Regionalna diferenciacija, vključno s speciacijo, ki je lahko vsaj deloma povezana s podzemeljsko fazo, naj bi se zgodila med zgodnjim in srednjim pleistocenom. Ocenjujemo, da se je začela invazija večine proučevanih živalskih linij v podzemlje Dinarskega krasa v obdobju med dvema in petimi milijoni let. Ključne besede: podzemlje, molekularna ura, molekularna flogenija, flogeografja, Dinarski kras.
INTRODUCTION
Te use of new molecular and systematic techniques us-ing allozymes and DNA sequences has enabled us to see a new picture of the evolution and diversity of subter-ranean fauna (e.g. Avise & Selander 1972; Sbordoni et al., 2000; Caccone & Sbordoni 2001; Leys et al., 2003; Verovnik et al., 2004; Gorički & Trontelj 2006; Lefébure et al., 2006; Zakšek et al., 2007). Molecular clock ap-
proaches should, at least in theory, enable us to date, to verify or to falsify previous hypotheses about the age of subterranean species. To be exact, it is usually not the age of a lineage or a taxon itself that is of special inter-est or under dispute, but the time since it has attained its subterranean nature, making it even more challeng-ing. Hypotheses and models explaining cave invasions
1 Oddelek za biologijo, Biotehniška fakulteta, Univerza v Ljubljani, Večna pot 111, 1000 Ljubljana, Slovenia, fax: +386 1 2573390, e-mail: peter.trontelj@bf.uni-lj.si
2 Notranjski muzej Postojna, Ljubljanska 10, 6230 Postojna, Slovenia.
Received/Prejeto: 30.01.2007
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PETER TRONTELJ, ŠPELA GORIČKI, SLAVKO POLAK, RUDI VEROVNIK, VALERIJA ZAKŠEK & BORIS SKET
and speciation in caves are well-elaborated (e.g. Rouch & Danielopol 1987; Holsinger 2000; Trajano 2005) and should thus ofer good grounds for the timing of such events and for testing their correlation with geographical, geological and hydrographical counterparts. For exam-ple, Leys et al., (2003) have shown that all evolutionary transitions to subterranean life in Australian dytiscids took place during the Late Miocene and Early Pliocene as a result of aridifcation. However, reliable data on the age of these events is surprisingly scarce. when such data are available, the accuracy is ofen below that of molecular clock rates. In fact, the use of molecular dating methods itself has introduced considerable uncertainty about how old subterranean species might be. while the youngest estimation, based on “classical” biological reasoning, is no more than 10,000 years (Sket 1997), the upper limit for the divergence of two subterranean sister species has been pushed to an incredible 110,000,000 years (Buhay & Crandall 2005).
Boutin and Coineau (2000) have argued that dating of cladogenetic events by a molecular clock is particular-
ly useful in the case when the dates are corroborated by other methods. Since the obvious problem of the Dinaric Karst area is that reliable dating for clearly defned vicari-ant events or the age of available subterranean habitat is lacking, it has been impossible to corroborate molecu-lar clock divergence by independent data. In this case a comparative phylogeographic approach might provide the means for an independent validation of age esti-mates. Comparative phylogeography seeks, as does his-torical biogeography, concordant geographical patterns of codistributed lineages (e.g. Arbogast & Kenagy 2001). Te evolution of codistributed phylogeographic groups of diferent taxa is likely to have been driven by the same historical factors, like vicariant events or climatic shifs.
In this contribution we (1) identify common phylo-geographic patterns among those troglobiotic taxa from the Dinaric Karst for which such data are available, and (2) estimate the timeframe of the corresponding cladoge-netic events using a global molecular clock approach.
MATERIAL AND METHODS
Te presented data were taken from several phylogeo-graphic studies of subterranean animals in the Dinaric Karst, including the ubiquitous aquatic isopod Asellus aquaticus Linne (Verovnik et al., 2004, 2005), the cave salamander Proteus anguinus Laurenti (Gorički 2006, Gorički & Trontelj 2006), and the cave shrimp troglo-caris s. lato (Zakšek et al., 2007). Further, we included unpublished sequences from studies that are in progress, including leptodirine cave beetles and aquatic sphaero-matid isopods from the genus monolistra. Te age esti-mations for the last two groups should be regarded as preliminary because in-depth analyses of phylogenetic relationships and corroboration by further loci are still under way. we were only interested in a small number of well-supported splits and therefore used straightfor-
ward minimum evolution searches with bootstrapping as implemented in MEGA (Kumar et al., 2004). Divergence time estimates are based on available clock-rate data for groups that are as closely related as possible (Caccone & Sbordoni [2001] for leptodirines, Ketmaier et al., [2003] for Asellus aquaticus, and Sturmbauer et al., [1996] and Schubart et al., [1998] for monolistra). To assure compat-ibility between molecular divergences we used the same models as were used in the original works describing the rates (Tamura-Nei distances with a gamma distributed rate variation among sites). where more than one hap-lotype per population or lineage was analyzed we used net between group distances to correct for ancestral in-traspecifc diversity.
RESULTS
Te split between major geographically defned lineages Te geographical distribution of troglobiotic (in-cluding stygobiotic) sister taxa can be used to infer inde-pendent cave invasions. For example, if the present-day ranges of two troglobionts are separated by large areas of non-karstic terrain without hypogean habitat, we can
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postulate an epigean last common ancestor. Examples of that kind can be found in the shrimp genus troglo-caris, with the hercegovinensis lineage inhabiting Trans-caucasian and SE parts of the Dinaric Karst where it is sympatric with the SE populations of the Anophthalmus lineage (Zakšek et al., 2007). Teir split estimated at 6–11
AGE ESTIMATES FOR SOME SUBTERRANEAN TAxA AND LINEAGES IN THE DINARIC KARST
Myr ago is the oldest, although unlikely, possible time of cave invasion. Te youngest split that could be reliably inferred from the phylogenetic tree and probably still oc-curred in surface waters, was the one between the Bos-nian lineage and other “Anophthalmus” lineages. Because the karst area in Bosanska Krajina, to which the Bosnian clade is restricted, is so remote and isolated from the rest of the Dinaric populations, it is reasonable to assume that an underground connection between them could never have existed. Te estimated time of this split, 3.7–5.3Myr ago, is hence the oldest possible age at which troglocaris anophthalmus might have invaded the Dinaric Karst underground (Tab. 1).
For the cave salamander Proteus anguinus, exhibit-ing a distribution pattern similar to that of troglocaris, the corresponding age of the Bosanska Krajina lineage was estimated at 4.4–5.4 Myr (Gorički 2006). However, older lineages exist that, theorethically, might have in-vaded caves even as early as 8.8–16 Myr ago (see also Fig. 1).
Another troglobiotic group restricted to the Dinaric Karst area and having a non-troglomorphic sister group is the Dinaric clade of Asellus aquaticus (see Verovnik et al., 2005). Te time of this split, and hence the maximum possible age of cave invasion is 3.8–4.8 Myr.
TIMING OF MORPHOLOGICAL CHANGES
where possible, we tried to combine the biology (e.g. degree of troglomorphism, lack of gene fow) of taxa with corresponding data on paleogeography and paleo-hydrography to infer speculative scenarios on how and when lineages might have switched to subterranean life and evolved troglomorphic traits. For example, we have some indication about how long at most it takes a salamander population to become troglomorphic. Since the subspecies P. a. parkelj Sket et Arntzen has retained its ancestral, non-troglomorphic characteristics, it is reason-able to conclude that its sister lineage must have evolved
troglomorphoses independently from other, less related troglomorphic lineages (Sket & Arntzen 1994; Gorički & Trontelj 2006; see Fig. 1). Te split between the non-troglomorphic lineage and its last troglomorphic sister lineage was estimated at 0.5–0.6 Myr based on mitochon-drial rDNA sequences, 1.1–2.4 Myr based on the mtDNA control region (Gorički 2006), and at 1.1–4.5 Myr by an allozyme clock (Sket & Arntzen 1994).
Asellus aquaticus has evolved several separate sub-terranean and troglomorphic populations. One of them, from the subterranean Reka River below the Kras/Carso Plateau, is genetically completely isolated from epigean populations at the Reka resurgence while there are no epi-gean populations in the Reka before the sink (Verovnik et al., 2003, 2004, 2005; Fig. 2). Further, it has no mtDNA
tab. 1. Estimated time (in million years) of some keystone events in the evolution of troglobionts in the dinaric Karst.
Taxon	Age of holodinaric group	Age of merodinaric group	Mid-Dinaric split	Northwest split
Troglocaris (Dinaric and Caucasian lineages)1	7.9–15.1	n.a.	n.a.	n.a.
Troglocaris anophthalmus agg.1	n.a.	3.7–5.3	1.3–2.3	1.5–2.1
Troglocaris hercegovinensis agg.1	n.a.	3.8–4.8	n.a.	n.a.
Proteus anguinus2	8.8–16.0	n.a.	8.8–16.0	4.2–5.2
Asellus aquaticus (Dinaric clade)3	n.a.	3.8–4.8	n.a.	0.8–1.2
Microlistra4	n.a.	1.1–2.3	n.a.	n.a.
Pseudomonolistra hercegoviniensis4	n.a.	0.3–1.0	n.a.	n.a.
Monolistra caeca4	n.a.	1.8–3.7	n.a.	n.a.
Leptodirus hochenwartii hochenwartii et L. h. reticulatus5	n.a.	1.9–2.0	n.a.	n.a.
1Using COI clock for shrimps (see Knowlton & Weigt 1998; zakšek et al., 2007)
2Using 12S and 16S rdNA clock for Newts (see Cacconesee et al., 1997; Gorički 2006)
3Using COI clock for subterranean Asellota (see Ketmaier et al., 2003; verovnik et al 2005)
4Using 16S r-RNA clock for fddler crabs (Sturmbauer et al 1996) and land crabs (Schubart et all 1998)
5Using COI clock for subterranean leptodirine beetles (Caccone & Sbordoni 2001)
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Fig. 1: A simplifed view of the phylogenetic relationships obetween troglomorphic and non-troglomorphic Proteus anguinus populations (from Gorički and trontelj 2006). Postulating a non-troglomorphic ancestor and unidirectional evolution toward troglomorphism, we can take the split between the black subspecies (non-troglomorphic) and its unpigmented sister lineage to estimate the maximal time (t1) needed for a salamander lineage to evolve the entire array of cave-related traits known in this taxon. If one accepts the notion of multiple independent cave invasions for Proteus, than t2 is the potentially oldest time since it has become subterranean.
and nuclear rDNA haplotypes in common with hypoge-an populations from the Ljubljanica River drainage with which the Reka drainage has been connected many times during the Pleistocene and occasionally even nowadays (Habič 1989). It is thus reasonable to assume that the an-cestor of the subterranean Reka River population invad-ed hypogean waters and became cave-adapted before any secondary contact could occur. Te estimated age of the Reka River lineage is 3.1–4.1 Myr (Verovnik et al., 2004), making it a pre-Pleistocene troglobiotic relict (Verovnik et al., 2004).
monolistra, a troglobiotic group of freshawater sphaeromatid isopods, shows a high taxonomic and mor-phological diversity restricted to the Dinaric Karst and parts of the Southern Calcareous Alps. According to our preliminary results of a molecular phylogenetic analysis based on nuclear and mitochondrial DNA sequences, there are at least three well-supported monophyla. Tese are the subgenus m. (microlistra), m. (monolistra) caeca Gerstaecker, and the polytypic m. (Pseudomonolistra) hercegoviniensis Absolon. Several lines of evidence sug-gest that the common ancestors of each of these groups invaded cave waters polytopically (Sket 1986, 1994). while we remain ignorant about when and how ofen an-cestral monolistra lineages invaded subterranean waters, we can expect that the radiation of at least some of the three groups took place in the underground. Teir ages
VALERIJA ZAKŠEK & BORIS SKET
Fig. 2: Te case of troglomorphic and non-troglomorphic lineages of Asellus aquaticus in the dinaric Karst, highly simplifed (from verovnik et al 2004, 2005). Te Reka and the Ljubljanica (Ljub) basin lineages have independently invaded subterranean waters and thus constitute separate taxa, although traditionally assigned to the same subspecies, A. a. cavernicolus. Te subterranean Reka River population presents the oldest stygobiotic lineage of Asellus aquaticus. because it is genetically completely distinct, it must have escaped interbreeding during various times of hydrological contact with surface populations. We therefore believe that it became a specialized stygobiont soon afer the split at time t1. Te Ljub lineage from the subterranean Ljubljanica River, although morphologically distinct, is still sharing mtdNA haplotypes with surface populations and thus represents a younger invasion. Eur and din denote various epigean European and dinaric lineages, respectively.
(maximally 0.4–3.7 Myr) give us an idea for how long some monolistra lineages have been dwelling in the Di-naric Karst underground.
Leptodirus hochenwartii Schmidt, a highly troglo-morphic leptodirine cave beetle, is the only terrestrial Dinaric troglobiont with available molecular dating. Us-ing a leptodirine COI clock calibration by Caccone and Sbordoni (2001) we estimated the age of the Leptodirus lineage by dating the split with Astagobius angustatus Schmidt, its slightly less troglomorphic sister lineage. Te estimated time of this split (8.7–9.8 Myr ago) is the oldest possible age at which the extremely specialized morphology of Leptodirus could have started evolving. Moreover, taking into account recent unpublished phy-logenetic fndings based on nuclear and mitochondrial gene sequences, the traditional subspecies of Leptodirus in fact represent distinct lineages with divergences well in the range of between species comparisons. Tese lineages all share the same constructive apomorphic troglomor-phic characters, and it seems probable these troglomor-phies have already existed at least at the time of their last common ancestor. Te time of divergence between basal Leptodirus lineages hence represents the youngest pos-sible age at which Leptodirus has evolved its full array
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of troglomorphic characters. Based on a yet incomplete taxonomic sample (L. h. hochenwartii Schmidt and L. h. reticulatus J. Müller) we tentatively dated it at 1.9–2.0 Mya.
TIMING OF PALEOHyDROGRAPHIC CHANGES
For some stygobiotic taxa with a broader Dinaric range, we identifed two concordant geographic patterns pos-sibly pointing to common underlying historical events, like changes in hydrographic connections. Tese vicari-ant patterns include (1) a split between a northwestern and southeastern Dinaric clade (mid-Dinaric split), and (2) a younger subdivision of the northwestern clade (or of a part thereof) into a western and eastern Slovenian lineage (Tab. 1).
It has been stated that some stygobiotic species inhabit areas that are hydrographically fragmented. Te
most parsimonious explanation of such distributions is that their ranges were hydrographically interconnected in the past. Tis may as well include the surface paleohy-drography that was heavily fragmented by karstifcation. Te (polytopic) immigration underground could thus have proceeded simultaneously with the separation of ancestral populations. we can illustrate this scenario by the case of some monolistra lineages, namely of the subgenus microlistra and of the species m. (m.) caeca. Some ten microlistra spp. are perfectly allopatric in distribution, mainly bound to actual watersheds. Another group, m. caeca, inhabits at least three watersheds, in which four named subspecies have evolved. According to a 16S rDNA molecular clock (Sturmbauer et al., 1996, Schubart et al., 1998), the system began to fragment about three million years ago.
DISCUSSION
Before we reach any conclusions we would like to note that dating of keystone events in the evolution of sub-terranean life, as well as anywhere else in evolution (e.g. Graur & Martin 2004), remains a highly speculative enterprise. Of central concern should be the fact that we are relying on a more or less global clock within certain taxonomic boundaries. Tese clocks usually rely on single calibration points (e.g. the separation of the Sardinia-Corsica microplate from the Iberian Peninsula; Ketmaier et al., 2003) and have mostly not been tested against in-dependent geological events.
Further, all our timings assume linear accumulation of substitutions over time, i.e. the existence of a valid mo-lecular clock. Although we can be quite sure that this as-sumption is violated to a certain extent, we can mitigate the problem by excluding those taxa from the analysis that violate the linearity assumption most. More sophisticated and realistically modeled approaches use a relaxed clock allowing for diferent local rates on diferent branches of the tree (e.g. Sanderson 2002). However, with single cali-bration points only, such approaches yield quite hopeless and certainly unrealistic intervals. For example, the age of the deepest split in the Niphargus virei (subterranean amphipod from France) complex was estimated at 14–19 Myr using a global Stenasellus clock, whereas the relaxed clock estimate was 22–71 Myr (Lefébure et al., 2006).
Tird, it should be noted that even with the aid of molecular phylogenetic tools the timing is still suscep-tible to incorrect estimations of relationships and incom-plete taxonomic coverage. For example, the timing of the
origin of the highly troglomorphic morphologies in Lep-todirus depends on the most basal split in the taxon. By not having included all known subspecies, we are facing the risk that some other subspecies might have branched of earlier than the studied ones.
One potentially useful way to improve our informal confdence in the timing of evolutionary events in subter-ranean animals is to look for phylogeographic correspon-dence of timings derived from independent taxa with in-dependent molecular clocks. At the present stage of most of our analyses such comparisons can only be preliminary. we can nevertheless notice that specifc groups of events belong to diferent age classes, most markedly the gap be-tween the age of holodinaric troglobionts and those with narrower distributions within the Dinaric Karst (Tab. 1). Te recent lineages of Proteus and troglocaris probably both originate from the Miocene Dinaride Lake System (Krstić et al., 2003), and the age of both taxa refects their diferentiation long before they invaded the hypogean environment (Sket 1997; Gorički 2006; Zakšek et al., 2007). Regional diferentiation, including speciation, which can at least in part be associated with a subterranean phase, appears to be much younger, ranging from Pliocene to mid-Pleistocene. Based on these estimates plus the esti-mated age of the Reka River lineage of Asellus aquaticus (see above) we, tentatively, suggest two to fve million years as the time when most of the analyzed lineages started in-vading the Dinaric Karst underground.
Te mid-Dinaric split of Proteus and troglocaris pthalmus does not seem to originate from the same
ano
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vicariant event as the latter was estimated to be younger by an order of magnitude. Another commonality of the phylogeographic pattern, the division between a western and an eastern clade in the Slovenian Dinaric Karst, might have a common hydrogeological cause in two sty-gobiotic crustaceans (A. aquaticus and t. anophthalmus) somewhere in the middle of the Pleistocene. In Proteus, however, the same split appears to be substantially older. In the Dinaric Karst we were, so far, unable to fnd reliable time estimates for paleogeographic events to cali-brate local molecular clocks in diferent lineages. Con-
versely, the timing of phylogenetic events can serve, inas-much as we rely on global molecular clocks, to estimate the date of geographical, hydrographical, and geological changes (Sket 2002). Te comparative phylogeographic approach and the use of diferent independent molecular clocks have enabled us for the frst time to propose a tim-escale for the evolution of troglobionts that is relatively consistent over a wide taxonomic range. Tis timescale is a preliminary one, though. we expect it to change with the inclusion of further taxa, the study of more genes and the use of more accurate molecular dating approaches.
ACKNOwLEDGMENTS
we are indebted to many friends and colleagues who      several research projects funded by the Slovenian Re-helped us with the acquisition of biological samples and      search Agency, and of the contract n° EVK2-CT-2001-accompanied us during feld work. we thank Gregor      00121- PASCALIS of the Fifh Research and Technologi-Bračko and Jožica Murko-Bulič for their indispensable      cal Development Framework Program supported by the assistance in the lab. Te presented work is the result of      European Community
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