Cold tolerance in terrestrial invertebrates inhabiting subterranean habitats


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Cold tolerance in terrestrial invertebrates inhabiting subterranean habitats

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Title:
Cold tolerance in terrestrial invertebrates inhabiting subterranean habitats
Series Title:
International Journalof Speleology
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Novak, Tone
Nina Å ajna
Estera Antolinc
Saška Lipovšek
Dušan Devetak
Franc Janžekovič
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English

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Subjects / Keywords:
Cold Resistance ( local )
Slovenia ( local )
Trogloxenes ( local )
Troglophiles ( local )
Troglobionts ( local )
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serial ( sobekcm )

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Abstract:
Most organisms are able to survive shorter or longer exposure to sub-zero temperatures. Hypothetically, trogloxenes characterized as not adapted, and troglophiles as not completely adapted to thermally stable subterranean environment, have retained or partially retained their ability to withstand freezing, while most troglobionts have not. We tested this hypothesis experimentally on 37 species inhabiting caves in Slovenia, analyzing their lower lethal temperatures in summer and winter, or for one season, if the species was not present in caves during both seasons. Specimens were exposed for 12 hrs to 1°C-stepwise descending temperatures with 48 hr breaks. In general, the resistance to freezing was in agreement with the hypothesis, decreasing from trogloxenes over troglophiles to troglobionts. However, weak resistance was preserved in nearly all troglobionts, which responded in two ways. One group, withstanding freezing to a limited degree, and increasing freezing tolerance in winter, belong to the troglobionts inhabiting the superficial subterranean habitats. The other group, which equally withstand freezing in summer and winter, inhabit deep subterranean or other thermally buffered subterranean habitats. Data on cold resistance can thus serve as an efficient additional measure of adaptation to particular hypogean environments.
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International Journalof Speleology, Vol. 43, no. 3.

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K26-05196 ( USFLDC: LOCAL DOI )
k26.5196 ( USFLDC: LOCAL Handle )

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International Journal of Speleology 43 (3) 265-272 Tampa, FL (USA) September 2014en-US*tone.novak@uni-mb.simorphological adaptation and do not complete their life cycle there. 2) Troglophiles alternate between the epigean and hypogean habitats or live permanently in subterranean habitats; they show some moderate adaptation, such as partly reduced eyes and adaptations to compensate for the lack of visual orientation. Some among these do not complete their life cycle underground, while others do. 3) Troglobionts complete their life cycle in a hypogean environment, and most of them clearly show troglomorphic characteristics, like eyelessness and depigmentation. In contrast to the frequent preference for these three ecological groups, Novak et al. (2012) found that, on the one hand, trogloxenes and troglophiles together represent a group of variously adapted species, rather than two ecologically clearly separated categories, and, on the other hand, troglobionts divide into two strictly separated subgroups. Invertebrates are ectothermal and at their critical thermal minimum they enter chill-coma, where neuromuscular transmission and movement cease INTRODUCTIONSoon after the description of the leiodid beetle Leptodirus hochenwartii Schmidt, 1832 from the cave discussion began on the adaptation and ecology of animal species in the subterranean environment. Many ecological classifications of these organisms have been proposed, each of them being in particular restrained by the limited knowledge of species ecophysiology (Sket, 2008), and most of them burdened with the author’s subjective judgments. Though not universally accepted (Desutter-Grancolas, 1999), the classification established by Schiner (1854) and Racovitza (1907) is sufficiently informative for many purposes in subterranean ecology and evolution (Boutin, 2004). It considers the three main ecological groups of animals in hypogean habitats: 1) Trogloxenes are the least adapted for living in the subterranean environment; they exhibit no Most organisms are able to survive shorter or longer exposure to sub-zero temperatures. Hypothetically, trogloxenes characterized as not adapted, and troglophiles as not completely adapted to thermally stable subterranean environment, have retained or partially retained their ability to withstand freezing, while most troglobionts have not. We tested this hypothesis experimentally on 37 species inhabiting caves in Slovenia, analyzing their lower lethal temperatures in summer and winter, or for one season, if the species was not present in caves during both seasons. Specimens were exposed for 12 hrs to 1C-stepwise descending temperatures with 48 hr breaks. In general, the resistance to freezing was in agreement with the hypothesis, decreasing from trogloxenes over troglophiles to troglobionts. However, weak resistance was preserved in nearly all troglobionts, which responded in two ways. One group, withstanding freezing to a limited degree, and increasing freezing tolerance in winter, belong to the troglobionts inhabiting the superficial subterranean habitats. The other group, which equally withstand freezing in summer and winter, inhabit deep subterranean or other thermally buffered subterranean habitats. Data on cold resistance can thus serve as an efficient additional measure of adaptation to particular hypogean environments. Cold resistance; Slovenia; trogloxenes; troglophiles; troglobiontsReceived 24 October 2013; Revised 26 March 2014; Accepted 7 April 2014 tolerance in terrestrial invertebrates inhabiting subterranean habitats. International Journal of en-USCold tolerance in terrestrial invertebrates en-US en-US inhabiting subterranean habitatsTone Novak1*, Nina ajna1, Estera Antolinc2, Saka Lipovek1,3, Duan Devetak1, 1en-US1en-USDepartment of Biology, Faculty of Natural Sciences and Mathematics, University of Maribor, Koroka cesta 160, SI-2000-Maribor, Sloveniaen-US2en-USElementary School, Ptujska cesta 30, SI-3252 Rogatec, Sloveniaen-US3en-USDepartment of Cell Biology, Faculty of Medicine, University of Maribor, Slomkov trg 15, SI-2000-Maribor, SloveniaAbstract: Keywords: Citation:

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266 International Journal of Speleology, 43 (3), 265-272. Tampa, FL (USA) September 2014 Novak et al.(Bowler & Terblanche, 2008; MacMillan & Sinclair, 2011). After thawing, they reactivate from this die. During late spring and early summer frosts, such recoveries or deaths are also usual in some species which periodically inhabit caves (Novak et al., 2004). One possible measure to determine cold resistance is the supercooling point (SCP), which is the lowest temperature an animal reaches before freezing (Lee, 2010); however, the ecological validity of the SCP may sometimes be ambiguous (Renault et al., 2002). Lower lethal temperatures (LLTs) are a more authentic measure of survival ability, usually expressed as LT50, which is the measure of temperature at 50% mortality of individuals exposed to temperatures below 0ar-SA C (Leather et al., ar-SA 1995; Watanabe, 2002). Response to below-zero temperatures is one such additional measure that enables a comparative insight on the general scale. The scarcity of evidence about LLTs in terrestrial animal residents in caves (Sacharov, 1930; Kirchner, 1973; Pullin & Bale, 1986; Novak et al., 2004, 2007; Latella et al., 2008; Lencioni et al., 2010) does not provide consistent information on the topic, since these authors used a variety of experimental methods. Although not as stable as had previously been supposed, in the temperate zone, the temperature in the deep subterranean habitats like deep bedrock fissures and caves is close to the mean annual value in surface habitats (Luetscher & Jeannin, 2004; Culver & Pipan, 2009a, b). However, freezing is usually not expected in caves. Consequently, the highest tolerance to sub-zero temperatures is expected in the trogloxenes and diminishes over the troglophiles to the troglobionts, which are adapted to the deep subterranean habitats. In practice, some aquatic (Hervant & Mathieu, 1997; Issartel, 2007; Colson-Proch et al., 2009) and terrestrial troglobionts (Peck, 1974; Latella et al., 2008; Lencioni et al., 2010) clearly show cold resistance. On the other hand, most Antarctic and Alpine species, which in their microhabitats are thermally buffered at above-zero temperatures, are intolerant to freezing (e.g., Zettel, 2000; Block, 2002; Elster & Benson 2004; Lipovek et al., 2004; Novak et al., 2004). In this study, our aim was to establish to what model fits with the expected decreasing resistance to temperatures below 0ar-SA C in selected central European and Dinaric species inhabiting caves. For this purpose we measured their LLTs in winter and summer, to detect eventual seasonal differences in the response. We also hypothesized that, within these species, the range of response to under-zero temperatures is widest in trogloxenes and diminish over troglophiles to troglobionts. Additionally, we tested whether, according to the LLTs, the presence of two troglobiont groups (shallow and deep sensu Novak et al., 2012) could be detected.MATERIALS AND METHODSSample collection Specimens of a representative series of 37 species divided among trogloxenes, troglophiles and troglobionts (Table 1) were selected for measurement of their LLTs. They were collected from 14 caves and abandoned mines (in the following text: caves) in Slovenia at altitudes of 365 m. Besides the two gastropod species, all the others were arthropods. The specimens were collected during two critical periods: in summer or in winter, or in both seasons if specimens were present in caves. In Amilenus and both Gyas species, individuals of various stages were present in summer (adults) and winter (nymphs), while adults, older and the younger larvae of both Troglophilus species occurred synchronically in caves. and three individuals each of the protected species were collected for the investigation. For a few species, we used a smaller number of specimens due to collecting difficulties. In 54 ecologically investigated caves in Slovenia measured air temperature profiles (details in Novak, 2005; Novak et al., 2004, 2012) were used to characterize habitat temperatures of the species studied. For the habitats for each species, we calculated the mean, minimal and maximal temperatures (Tmean, Tmin and Tmax) to compare with the LLTs. Laboratory analyses The following protocol was arranged to measure LLTs and simulate repeated frosts in natural habitat. The LLT measurements were carried out in a precise thermostatic cooling chamber THK/V1 (Elpromer, Slovenia) with a temperature adjustment of .1C, and a cooling/warming movement of ~10C/hr. Measurements started at -2.0C and were stopped at -12.0C. Experimental individuals wet paper in 2 dL vessels moistened every 5th day to prevent desiccation. After a two-day acclimation in a refrigerator at 6.0C, the vessels were placed in the cooling chamber. The specimens were exposed to a particular temperature for 12 hrs, afterwards kept for 48 hrs in the refrigerator at 6.0C and then exposed to a temperature 1.0C lower. Specimens’ condition was checked after 24 hrs at 6.0C, when the eventual dead ones were removed. The procedure was repeated until all the specimens had died. Statistical analysis Specimens differing by sex and stage were first tested using the Mann-Whitney U test with a Bonferroni correction for two comparisons, and in Troglophilus neglectus where adults, older and younger larvae co-occurred, for six comparisons to analyze whether they differ in LLTs either in summer or winter (in the following: s and w). There were no significant differences in any experimental species (adjusted p value for sex: p>0.025, for T. neglectus: p>0.008)

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267 International Journal of Speleology, 43 (3), 265-272. Tampa, FL (USA) September 2014Cold-hardiness in subterranean invertebratesin either s or w season, allowing the combining of all specimens of a species irrespectively of sex and developmental stage in further analyses. For the analysis of trends in cold resistance, the species mean seasonal LLTs were calculated. For 28 species with both the s and the w LLT measurements, a normalized plot of the s vs. the w LLTs was used for graphical analysis of species pre-classified within trogloxenes, troglophiles and troglobionts (Table 2). Seasonal differences among categories in their mean LLTs were tested separately for s and w using analysis of variance (ANOVA), followed by a post-hoc mean separation by the Tukey Honest Significant Difference (HSD) test for unequal sample sizes. The effect size was calculated using the formula (Field, 2009). To demonstrate general trends in the response differences among trogloxenes, troglophiles and troglobionts, we calculated the integrated distributional curves of the s and w LLTs for each category. To evaluate the possible presence of two categories of troglobionts (Novak et al. 2012), if such two groups exist also according to their s and w LLTs, we grouped Ischyropsalis hadzii, Anophthalmus hitleri , Ceuthmonocharis pusillus, and C. robici robici as previously recognized shallow subgroup and Andronuscus stygius, Titanethes albus, Aphaobiella tisnicensis, and Leptodirus hochenwartii as deep subgroup for further detailed comparisson. The agreement of the LLTs with air Tmean, Tmin and Tmax at the settled sites within the 54 caves was tested using the correlation analysis. The data analysis was carried out with the statistical software SPSS 21.0.RESULTSAmong the 37 species under study, 28 were collected from caves and tested for LLTs in s and w, five only in s and four only in w (Table 2). Seasonal differences in LLTs Seasonal differences between s and w LLTs of individual species varied from non-significant to highly significant (Table 2). Most species could equally withstand s and w LLTs, while a few were more resistant to s than to w freezing, and the reverse for a few other species. On average, the LLTs differed by less than 1C between the seasons. Only a few s and the w LLTs (Table 3). Within the groups, the s and w LLTs did not significantly differ in trogloxenes, but differed significantly in troglophiles ( Mann-Whitney U test, U=1269.5, p=0.001 ) and troglobionts (Mann-Whitney U test, U=842.5, p<0.001). Their corresponding effect sizes were 0.045, 0.289 and 0.363, respectively, i.e., increasing from non-existent in trogloxenes over small in troglophiles to medium in troglobionts. Differences in LLTs between the ecological groups The LLT range was the widest for the trogloxenes (sar-SAC; w troglophiles (s w the troglobionts (s w freezing decreased from trogloxenes over troglophiles to troglobionts (Fig. 1). We obtained a statistically significant overall F test from the ANOVA for the mean LLTs in s (F2,30=6.97, p=0.003). Posteriori testing showed significant differences between trogloxenes and troglobionts (HSD for unequal N, p=0.017). Normalized results of the LLTs are shown in Fig. 2 together with the best fits of error function (Gauss error function) to the data. Subgroups in troglobionts All troglobionts under study exposed a certain degree of freezing tolerance, i.e., they withstood temperatures below 2C. A t test showed no significant difference between the s and the w LLTs in trogloxenes and troglophiles, while troglobionts demonstrated significantly lower LLTs in w (t=4.26, df=106, p<0.001). The troglobionts classified in the shallow en-USHigher taxonen-US Familyen-US Speciesen-US Gastropodaen-US Helicidaeen-US 1 x en-USChilostoma (Josephinella) lefeburianaen-US en-US en-US en-US 2 x en-USFaustina illyricaen-US (Stabile 1864)en-US Oniscoideaen-US Trichoniscidaeen-US 3 t en-US Andronuscus stygiusen-US en-US 4 t en-US Titanethes albusen-US (C. Koch 1841)en-US Araneaeen-US Agelenidaeen-US 5 x en-USMalthonica silvestrisen-US (L. Koch 1872)en-US Linyphiidaeen-US 6 f en-US Troglohyphantes diabolicusen-US en-US en-US en-US Nesticidaeen-US 7 x en-USNesticus cellulanusen-US (Clerck 1757)en-US Tetragnathidaeen-US 8 f en-US Meta menardien-US en-US en-USMetellina merianaeen-US (Scopoli 1763)en-US Opilionesen-US Sclerosomatidaeen-US en-USAmilenus aurantiacusen-US (Simon 1881)en-US 11 x en-USGyas annulatusen-USen-US 12 x en-USGyas titanusen-USen-US 13 x en-USLeiobunum rupestreen-US en-US Ischyropsalididae en-US 14 t en-USIschyropsalis hadziien-US en-US Nemastomatidaeen-US 15 x en-USParanemastoma bicuspidatumen-US en-US en-US (C. L. Koch 1835)en-US 16 x en-USParanemastoma quadripunctatumen-US en-US en-US (Perty 1833)en-US Microcoryphiaen-US Machilidaeen-US 17 x en-USTrigoniophthalmus alternatusen-US en-US Orthopteraen-US Rhaphidophoridaeen-US 18 f en-USTroglophilus cavicolaen-US (Kollar 1833), en-US en-US w: adults + larvaeen-US en-USTroglophilus neglectusen-US en-US en-US w: adults + larvaeen-US Lepidopteraen-US Geometridaeen-US en-USTriphosa dubitataen-US (Linnaeus 1758)en-US Noctuidaeen-US 21 x en-USScoliopteryx libatrixen-US (Linnaeus 1758)en-US Nymphalidaeen-US 22 x en-USAglais ioen-US (Linnaeus 1758)en-US Coleopteraen-US Carabidaeen-US 23 t en-USAnophthalmus hitlerien-US en-US 24 f en-USLaemostenus (en-US Antisphodrusen-US ) schreibersiien-US en-US en-US (Kster 1846) en-US Leiodidaeen-US 25 t en-USAphaobiella tisnicensisen-US en-US 26 t en-USAphaobius milleri alphonsien-US en-US 27 x en-USCatops tristisen-USen-US 28 t en-USCeuthmonocharis pusillusen-US en-US en-USCeuthmonocharis robici robicien-US en-US en-US en-US en-USCholeva sturmi en-US (Brisout 1863)en-US 31 t en-USLeptodirus hochenwartiien-US (Schmidt 1832)en-US Hymenopteraen-US Ichneumonidaeen-US 32 x en-USDiphyus quadripunctoriusen-US (Mller 1776)en-US Dipteraen-US Culicidaeen-US 33 x en-USCulex pipiensen-US (Linnaeus 1758)en-US Heleomyzidaeen-US 34 x en-USHeleomyza (en-US Heleomyzaen-US )en-US cf. en-US captiosaen-US en-US en-US en-US Limoniidae en-US 35 x en-USChionea (Sphaeconophilus) austriacaen-US en-US en-US en-US 36 x en-USLimonia nubeculosaen-US en-US Mycetophilidae en-US 37 f en-USSpeolepta leptogasteren-US (Winnertz 1863), larvaeen-USTable 1. Experimental species to determine LLTs. Biospeological en-US categories: x trogloxene, f troglophile, t troglobiont. en-US wen-US winter.

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268 International Journal of Speleology, 43 (3), 265-272. Tampa, FL (USA) September 2014 Novak et al. en-USSpeciesen-US Ns/Nwen-US sen-US-LLTsen-US Mean Sden-US Min – Maxen-US Ts cavesen-US Meanen-US Min – Maxen-US wen-US-LLTsen-US Mean Sden-US Min – Maxen-US Tw cavesen-US Meanen-US Min – Maxen-US Speciesen-US Ns/Nwen-US sen-US-LLTsen-US Mean Sden-US Min – Maxen-US Ts cavesen-US Meanen-US Min – Maxen-US wen-US-LLTsen-US Mean Sden-US Min – Maxen-US Tw cavesen-US Meanen-US Min – Maxen-US 1 en-USChilostoma en-US en-US lefeburianaen-US 5 / 5en-US *en-US en-US -4 – -3en-US 16.4en-US 15.1 – 17.6en-US en-US -5 – -4en-US en-US en-US en-USTriphosa dubitataen-US en-US **en-US -8.4 2.2en-US -11 – -5en-US 11.8en-US en-US en-US -5 – -4en-US 2.3en-US en-US 2 en-USFaustina illyricaen-US en-US 5 / 5en-US nsen-US en-US -5 – -4en-US 13.7en-US en-US en-US -5 – -4en-US 2.6en-US en-US 21 en-USScoliopteryx libatrixen-US en-US -en-US -8en-US 11.6en-US en-US en-US -7 – -5en-US 3.4en-US -3.2 – 11.4en-US 3 en-USAndronuscusen-US en-US stygiusen-US en-US nsen-US en-US -4 – -3en-US en-US en-US en-US -4 – -3en-US 8.5en-US 8.5 – 8.5en-US 22 en-USAglais ioen-US 2 / 3en-US nsen-US en-US en-US 11.6en-US 8.7 – 14.4en-US en-US en-US 5.2en-US 5.2 – 5.2en-US 4 en-USTitanethes albusen-US en-US nsen-US en-US -4 – -2en-US 8.6en-US 8.5 – 8.8en-US en-US -4 – -2en-US en-US en-US 23 en-USAnophthalmus hitlerien-US 2 / 2en-US -en-US en-US -3 – -3en-US 8.3en-US en-US en-US -5 – -4en-US 8.2en-US 8.1 – 8.2en-US 5 en-USMalthonica en-US en-US silvestrisen-US 6 / 4en-US nsen-US en-US -8 – -4en-US 11.7en-US en-US en-US -8 – -4en-US 2.2en-US en-US 24 en-USLaemostenus en-US en-US schreibersiien-US 3 / 8en-US nsen-US en-US -5 – -4en-US en-US 5.7 – 18.3en-US en-US -5 – -4en-US en-US en-US 6 en-USTroglohyphantes en-US en-US diabolicusen-US 7 / 4en-US nsen-US -7.6 1.6en-US en-US en-US 8.2 – 21.3en-US -7.8 1.3en-US en-US 5.7en-US en-US 25 en-USAphaobiella en-US en-US tisnicensisen-US 6 / 6en-US nsen-US en-US -3 – -3en-US en-US en-US en-US -4 – -3en-US 8.2en-US en-US 7 en-USNesticus cellulanusen-US en-US en-US nsen-US -7.3 1.3en-US en-US en-US 8.1 – 14.8en-US en-US en-US 6.2en-US en-US 26 en-USAphaobius milleri en-US en-US alphonsien-US / 4en-US -en-US en-US -4 – -3en-US 7.5en-US en-US 8 en-USMeta menardien-US en-US nsen-US en-US -8 – -2en-US 12.6en-US en-US -4.5 1.5en-US -8 – -3en-US 4.4en-US -4.5 – 11.4en-US 27 en-USCatops tristisen-US 4 / -en-US -en-US en-US -7 – -5en-US en-US en-US en-USMetellina merianaeen-US en-US en-US nsen-US -6.6 1.7en-US -8 – -3en-US 12.5en-US en-US -7.6 1.2en-US en-US 2.4en-US -3.6 – 11.4en-US 28 en-USCeuthmonocharis en-US en-US pusillusen-US 6 / 6en-US **en-US en-US -4 – -3en-US 13.3en-US 12.2 – 15.5en-US en-US -6 – -5en-US en-US en-US en-USAmilenus en-US en-US aurantiacusen-US en-US nsen-US en-US -5 – -4en-US 14.7en-US 7.2 – 21.5 en-USen-US -5 – -4en-US 5.5en-US -3.4 – 11.4en-US en-USC. robici robicien-US 6 / 6en-US **en-US en-US -4 – -3en-US en-US 5.7 – 14.8en-US en-US -5 – -4en-US en-US en-US 11 en-USG. annulatusen-US en-US ***en-US en-US -5 – -4en-US en-US en-US en-US -3 – -2en-US 5.7en-US 2.1 – 8.4en-US en-USCholeva sturmien-US / 2en-US -en-US en-US -4 – -4en-US 7.3en-US 7.3 – 7.7en-US 12 en-USGyas titanusen-US en-US nsen-US en-US -4 – -3en-US en-US en-US en-US -4 – -2en-US en-US -3.5 – 6.5en-US 31 en-USLeptodirus en-US en-US hochenwartiien-US 6 / 6en-US nsen-US en-US -5 – -3en-US 8.6en-US 8.5 – 8.8en-US en-US -5 – -4en-US en-US en-US 13 en-USLeiobunum en-US en-US rupestreen-US en-US -en-US en-US -4 – -3en-US 17.3en-US en-US 32 en-USDiphyus en-US en-US quadripunctoriusen-US / 2en-US -en-US en-US -8 – -7en-US en-US 2.6 – 8.4en-US 14 en-USIschyropsalis en-US en-US hadziien-US 6 / 6en-US **en-US en-US -2 – -2en-US 8.7en-US en-US en-US -4 – -3en-US 8.6en-US 8.6 – 8.7en-US 33 en-USCulex pipiensen-US 2 / 5en-US nsen-US en-US -6 – -5en-US 15.7en-US 8.5 – 21.5en-US en-US -5 – -5en-US 4.1en-US en-US 15 en-USParanemastoma en-US en-US bicuspidatumen-US en-US -en-US en-US -4 – -3en-US en-US 7.7 – 12.2en-US 34 en-USHeleomyza cf. en-US en-US captiosaen-US 5 / -en-US -en-US en-US -12 – -8en-US 8.3en-US en-US 5.2en-US 1.8 – 8.1en-US 16 en-US P. quadripunctatumen-US en-US *** en-USen-US -5 – -4en-US 11.2en-US en-US en-US -8 – -6en-US 35 en-USChionea austriacaen-US / 1en-US -en-US -7en-US 5.3en-US en-US 17 en-US Trigoniophthalmus en-US en-US alternatusen-US en-US ***en-US en-US -3 – -2en-US 8.1en-US en-US en-US -4 – -4en-US 4.1en-US en-US 36 en-USLimonia nubeculosaen-US en-US -en-US en-US -5 – -4en-US en-US en-US 18 en-USTroglophilus en-US en-US cavicolaen-US en-US **en-US en-US -4 – -3en-US 12.3en-US 7.6 – 21.5en-US en-US -6 – -4en-US en-US en-US 37 en-USSpeolepta leptogasteren-US en-US **en-US en-US -5 – -3en-US 11.1en-US en-US en-US -2 – -2en-US 3.1en-US -3.5 – 8.4en-US en-UST. neglectusen-US en-US *en-US en-US -4 – -3en-US 13.5en-US en-US en-US -6 – -4en-US 5.8en-US en-USTable 2. Descriptive statistics of LLTs and ambient air T in 37 terrestrial invertebrate species inhabiting caves. Current numbers before species en-US en-USsen-US, Nen-USwen-US number of individuals in summer and winter; en-US sen-US-LLTs, en-USwen-US-LLTs summer and winter LLTs, and Mann-en-US en-USsen-US-LLTs vs. en-US wen-USen-USsen-US caves and en-US Ten-USwen-US caves summer and winter temperature in cave sections where species occurred. -10 -8-6-4-20Winter mean LLT [C] -10 -8 -6 -4 -2 0Summer mean LLT [C] Trogloxenes Troglophiles Troglobionts 1 31 8,19 14 29 23 3 18 25 10 24 17 28 22 6 7 9 2 5 16 21 20 33 11 37 12 4 en-USen-US as in Table 2) with both summer and winter data. The dotted line en-US represents the balance axis between the summer and winter LLTs.subgroup, represented by Ischyropsalis hadzii, Anophthalmus hitleri, Ceuthmonocharis pusillus and C. robici robici , significantly enhanced their resistance to sub-zero temperatures in winter, lowering their w LLTs at an average of 1.5ar-SA C with respect to the s LLTs, while Andronuscus stygius, Titanethes albus, Aphaobiella tisnicensis and Leptodirus hochenwartii, classified in the deep subgroup, did not (Fig. 3). skThe species of sk the shallow troglobiont subgroup and the representatives of the deep troglobiont respectively. Between shallow and deep subgroup there was no statistical difference in s LLTs, while these differences were significant for w LLTs ( Mann-Whitney U test, Z=3.55, p<0.001). Correspondence between the LLTs and caves temperatures In all the species under study the LLTs were lower than Tmin in the cave placements where the specimens occurred. There were no correlations between the LLTs and Tmin, Tmean and Tmax and the placement of these specimens in the caves.

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269 International Journal of Speleology, 43 (3), 265-272. Tampa, FL (USA) September 2014Cold-hardiness in subterranean invertebrates2000; Block, 2002; Block & Zettel, 2003; Ansart & Vernon, 2003; Sinclair et al., 2003; Hodkova & Hodek, 2004; Danks, 2006; Lagerspetz & Vainio, 2006; Hawes & Bale, 2007; Bowler & Terblanche, 2008; MacMillan & Sinclair, 2011; Vesala & Hoikkala, 2011). The stepwise 1ar-SAC-graded arrangement of our experiment does not allow precise determination of the LLTs; the real values may be, on average, about 0.5ar-SAC higher. On the other hand , although frequent freezing and rethawing is not painful to insects, it does cause permanent injuries, which in turn lower their ability to sustain low temperatures (Marshall & Sinclair, 2011). Like SCPs, LLTs also depend on the duration of exposure to low temperatures; after long-term exposure, the organisms may die from the exhaustion of energy reserves (Renault et al., 2002). because of many repeated refreezing and rethawing during the experimental procedure (cf. Marshall & Sinclair, 2011). Gyas annulatus represents a special case among trogloxenes, showing no freezing tolerance in winter, which is the consequence of overwintering in water current-close, thermostated overwintering habitats (Novak et al., 2004). Speolepta leptogaster among troglophiles and Ischyropsalis hadzii among troglobionts are further cases of seasonal intolerance.DISCUSSIONMultiple freeze–thaw cycles in a single winter are common in surface habitats in temperate latitudes and may present significant challenges to survival in insect species (Marshall & Sinclair, 2011). While specimens in deep caves can effectively avoid extreme values, such conditions are expected widely in ice and other cold caves, the entrance cave sections and bedrock fissure systems connected with the surface and the SSHs (Gers, 1998; sk Culver & Pipan, 2009a, b; sk ). Most frequently subfreezing conditions occur in winter conditions without snow, which is often caused by wind, especially the strong north-east wind, the bora (burja), -1 (WineAndWeather, 2011) in the Dinaric region. The methodology used in our investigation simulated the repeated half-day frosts which occasionally affect the habitats of many species under study. Cold resistance varies between and within species and depends on many factors, such as the developmental stage and age of an individual, its genetic potential, the season, the chill-coma temperature and the duration of exposure to low temperatures, nutrition, cold hardiness dynamics itself, diapause dynamics, photoperiodism, exposure to air currents, cryo-protective dehydration, the presence of nucleation agents and nucleation mitochondrial disintegration, refreezing, experimental conditions etc., all these within the wide range of cryo-protection plasticity (Storey & Storey, 1988; Leather et al., 1995; Smme, 1999; Worland et al., -1 -8 -6 -4 -2 . .2 .4 .6 .8 1. meanS.D. Troglophiles-summer -3.81.5 Troglophiles-winter -4.6 .5 Troglobionts-summer -3.11. Troglobionts-winter -4. 1.4 Trogloxenes-summer -5.53.5 Trogloxenes-winter -5.13. Sumofsurvivedspecies[norm.] LLTs[oC] en-UStroglophiles and troglobionts exposed to 12-hr freezing intervals. The en-US curves represent best fits to the error function to the data. -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 3 25 31 4 23 14 28 2 .26C TW=TS-1.5C Summermean[C] Wintermean[C] en-USen-US subgroup, species 3, 4, 25, 31 belong to deep subgroup. The solid en-US line represents the balance axis between the summer and winter en-US LLTs and dotted lines are guide to the eye for winter mean LLTs (Ten-USWen-US), en-US which are lower than summer mean LLT (Ten-USSen-USen-US subgroup or 1.5C, respectively. en-USThe lowest Ten-US Trogloxenesen-US Troglophilesen-US Troglobiontsen-US in summeren-US 11 en-USGyas annulatusen-US (-2.1)en-US en-USTriphosa dubitataen-US en-US 21 en-USScoliopteryx libatrixen-US (-2.3)en-US 37 en-USSpeolepta leptogasteren-US en-US in winteren-US en-USMetellina merianaeen-US en-US 16 en-USParanemastoma quadripunctatumen-US (-3.1)en-US 17 en-USTrigoniophthalmus alternatusen-US (-1.3)en-US 8 en-USMeta menardien-US (-1.1)en-US 18 en-USTroglophilus cavicolaen-US en-US en-USTroglophilus neglectusen-US (-1.2)en-US 14 en-USIschyropsalis hadziien-US (-1.5) en-US 23 en-USAnophthalmus hitlerien-US (-1.5)en-US 28 en-USCeuthmonocharis pusillusen-US (-1.4)en-US en-USC. robici robicien-US (-1.6)en-USen-US brackets. Numbers in front of the names as in Table 2.

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270 International Journal of Speleology, 43 (3), 265-272. Tampa, FL (USA) September 2014 Novak et al. year. This subgroup appears to have adapted to the deep subterranean and any other subterranean habitat where freezing does not occur. Species carrying out its amphibious way of life, like Titanethes albus, known to submerge in water for long periods (Sket, 1986; Vittori et al., 2012), may avoid freezing in this way. Besides, species preferring skthe terrestrial phreatic environment sk (milieu phreatique terrestre sk sensu sk Jeannel, 1926), like Aphaobiella tisnicensis, are skthermally buffered at above-sk zero temperatures (Novak et al., 2012). Such intolerance to freezing in terrestrial animals has been reported also from other close-to-water subsurface habitats, e.g., sk beside glaciers (Zettel, 2000), and between pebbles and sk stones near water streams (Novak et al., 2004). CONCLUSIONSOur study reveals that in central European and Dinaric trogloxenes, troglophiles and troglobionts, resistance to temperatures below 0C generally decreases in accordance with their increasing adaptation to the hypogean habitat. Trogloxenes are most diverse in tolerance to sub-zero temperatures, from non-existent to high. Troglophiles are in between trogloxenes and troglobionts with this respect. Most troglobionts show moderate resistance to freezing, and are divided into the two identified ecological subgroups. Species of the first subgroup stand subzero temperatures significantly better in winter than preferred habitats. The second troglobiont subgroup responds more or less equally in summer and winter, which is considered a residual evolutionary tolerance. Both such responses appear also in troglophiles.ACKNOWLEDGEMENTSWe are indebted to four anonymous referees for insightful comments on the manuscript, and to Michelle Gadpaille for valuable improvement of the language and Andrej orgo for discussion on statistical issues. We thank the Slovenian Environment Agency at the Ministry of Agriculture and Environment for permission to collect a limited number of selected protected subterranean species. This study was partly supported by the Slovenian Research Agency within the Biodiversity Research Programme (Grant No. P10078) and Infrastructure Programs of the Slovenian Research Agency (IP-0552).REFERENCESAnsart A. & Vernon P., 2003 Cold hardiness in molluscs. Acta Oecologica, 24: 95-102. þ en-UShttp://dx.doi.org/10.1016/S1146-609X(03)00045-6 Block W., 2002 Interactions of water, ice nucleation and desiccation in invertebrate cold survival. European Journal of Entomology, 99: 259-266. þ en-UShttp://dx.doi.org/10.14411/eje.2002.035 Block W. & Zettel J., 2003 Activity and dormancy in relation to body water and cold tolerance in a winteractive springtail (Collembola) . European Journal of Entomology, 100: 305-312. þ en-UShttp://dx.doi.org/10.14411/eje.2003.049Despite these problems, the overall responses of the 37 selected central European and Dinaric trogloxenes, troglophiles and troglobionts are comparable on the general level: lower a species sub-zero LLT, desto greater variability in individual LLTs. The species under study represent the expected general response to freezing: there is a clear decline in their LLTs from trogloxenes, to troglophiles and troglobionts. This is congruent with the general hypothesis of their increasing adaptation to the habitat. Three troglophilic species deserve comment. The spider Troglohyphantes diabolicus is relatively common in the investigated caves (Novak et al., 2012), where individuals usually settle in wall fissures and recesses of up to 10 cm in diameter, and similar sized sites between stones and pits in the clay. For this reason, they frequently make webs over the pitfall trap orifices (authors and Slavko Polak’s unpublished data). It has also been reported from small mammal burrows (Deeleman Reinhold, 1978) and water drips, which probably originate in vertical bedrock fissures (Pipan et al., 2008). This species thus prefers narrow subsurface habitats rather than caves, which is in agreement with its relatively high level of freezing resistance throughout the year, comparable to that for the trogloxenes. A second troglophic spider species, Meta menardi, is better adapted to the hypogean habitat than the similarly sized trogloxene Metellina merianae (Novak et al., 2010) and its average LLT fits into the troglophile range, and a few individuals showed the relatively high freezing resistance of -8ar-SAC. Such tolerance has been previously reported by Turquin (1971). Being also reported from screes (sk ) we support sk that M. menardi is among species showing an intermediate stage in adaptation to the hypogean habitat. In contrast, Speolepta leptogaster larvae show no cold resistance in winter. They mostly settle on ceiling and upper walls in caves emitting warm air. Such cave ceiling and blow holes, inaccessible to humans, form another thermally stable subterranean microhabitat, where temperatures generally do not fall below 0ar-SAC during winter. Like G. annulatus, S. leptogaster show moderate cold resistance in summer. Nearly all troglobionts under study showed a weak tolerance to below-zero temperatures. This is in accordance with Peck’s (1974) and Leinconi et al. (2010) findings in other troglobionts. The investigated troglobionts responded in two ways and can be strictly differentiated with this respect. sk The species sk of the shallowsk troglobiont subgroup Ischyropsalis hadzii, Ceuthmonocharis pusillus, C. robici robici , and Anophthalmus hitleri freezing, enduring 1.5C lower mean temperatures, in average, in winter than in summer. This seasonality clearly indicates their response to the winter external conditions which temporarily disturb their habitats, such as SSHs, by winter frosts. Androniscus stygius, Titanethes albus, Aphaobiella tisnicensis and Leptodirus hochenwartii and nearly equal freezing resistance throughout the

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