Morphological and Genetic Differentiation of Eremina Desertorum (Gastropoda, Pulmonata, Helicidae) in Egypt

Zoologica Scripta

Morphological and genetic differentiation of Eremina desertorum (Gastropoda, Pulmonata, Helicidae) in Egypt

REHAM F. ALI,MARCO T. NEIBER,FRANK WALTHER &BERNHARD HAUSDORF

Submitted: 7 May 2015 Ali, R.F., Neiber, M.T., Walther, F. & Hausdorf, B. (2016). Morphological and genetic dif- Accepted: 29 June 2015 ferentiation of Eremina desertorum (Gastropoda, Pulmonata, Helicidae) in Egypt. —Zoologica doi:10.1111/zsc.12134 Scripta, 45,48–61. To understand the processes that result in morphological and genetic diversity, we studied the differentiation of the land snails Eremina d. desertorum and Eremina desertorum irregularis in the deserts of northern Egypt. These two taxa are differentiated with regard to shell size and shape and are separated by a narrow hybrid zone west of Alexandria. The lack of differences in the genitalia and the lack of reciprocal monophyly of the mitochondrial haplotypes of E. d. desertorum and E. desertorum irregularis support their classiﬁcation as subspecies rather than distinct species. Low genetic distances indicate that the differentiation is probably less than half a million years old. The genetic data indicate a population expansion in agreement with other evidence that the Nile region in northern Egypt was more humid well into historical times than today. Shell size and shape are correlated with a climatic gradient from cooler and more humid conditions along the Mediterranean coast to arid and hot conditions in the interior. The decrease of body size with decreasing precipitation and increasing temperature might be explained by limited time for food intake in the more arid regions. The shape differences between the taxa are partly an indirect consequence of selection for body size, but are also directly affected by selection for reduction of aperture size. Corresponding author: Bernhard Hausdorf, Zoological Museum, Centre of Natural History, University of Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany. E-mail: [email protected] Reham F. Ali, Department of Zoology and Agricultural Nematology, Faculty of Agriculture, Cairo University, Post Box 12613, Gammaa Street, Giza, Egypt. E-mails: [email protected] Marco T. Neiber, Frank Walther, and Bernhard Hausdorf, Zoological Museum, Centre of Natu- ral History, University of Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany. E-mails: [email protected], [email protected]

Introduction mental gradient of decreasing precipitation and increasing A central goal of evolutionary biology is the understanding temperature from the Mediterranean coast inland. of the processes that result in morphological and genetic Eremina Pfeiffer, 1855 includes large desert snails that are diversity. Land snails were often used as model organisms distributed through northern Africa from the Cape Verde for evaluating the roles of selection and history in the Islands, Mauretania and Morocco eastwards to the Sinai origins of biodiversity (Davison 2002). In particular notable Peninsula, the Gaza strip and southern Israel, and south- are the classical studies of Cepaea that nicely illustrated the wards to Somalia (Hesse 1915; Kaltenbach 1934, 1942; Lla- action and effects of natural selection on shell phenotypes bador 1960; Verdcourt 1960; Groh 2005; Heller 2009). The in the wild (Cain & Sheppard 1954). However, patterns of genus is represented by E. advena (Webb & Berthelot, 1833) diversity are often difﬁcult to detect and to understand if and E. myristica (Shuttleworth, 1852) (Groh 2005) on the they are inﬂuenced by a host of historical and environmen- Cape Verde Islands and by E. dillwyniana (Pfeiffer, 1851) tal factors in complex ecosystems (e.g. Fiorentino et al. (=Helix duroi Hidalgo, 1886 = E. linanprietoae Cossignani & 2008). Therefore, we have studied the morphological and Galindo, 2012) and E. vermiculosa (Morelet, 1874), of which genetic differentiation of the land snail Eremina desertorum E. inexpectata Llabador, 1960 is considered a subspecies (Forsskal, 1775) in a manageable ecosystem, the deserts of (Kittel 2012), in Northwest Africa (Llabador 1960). In northern Egypt, which is dominated by a steep environ- Northeast Africa, E. desertella (Jickeli, 1872) is found along

48 ª 2015 Royal Swedish Academy of Sciences, 45, 1, January 2016, pp 48–61 R. F. Ali et al. Differentiation in Eremina desertorum in Egypt the coast of the Red Sea from Sudan to Cape Guardafui in In this study, we examined the morphological and Somalia (Verdcourt 1960; records from northern Egypt genetic differentiation of the two main taxa of this (Pallary 1909) refer to small forms of E. desertorum) and is complex, E. d. desertorum (Fig. 1A–C) and E. desertorum perhaps introduced at the Red Sea coast of Arabia (Neubert irregularis (Figs 1D–E and 2), and its causes. Eremina d. de- 1998). The most diverse complex of Eremina taxa is found in sertorum differs from E. desertorum irregularis in several Egypt and adjacent Mediterranean regions, westwards characters (Kaltenbach 1934), but all show considerable through Libya to southern Tunisia and eastwards through variation within these taxa so that none of these characters the Sinai Peninsula to the Gaza strip and southern Israel. alone can be considered decisive. The shells of E. d. deser- Some authors included all these forms in a single, highly torum are usually smaller and more depressed than those of variable species, E. desertorum (e.g. Hesse 1915; Biggs 1959), E. desertorum irregularis. In the latter, the umbilicus is whereas others classify E. irregularis (Ferussac, 1821) (=Helix closed, whereas there is sometimes a narrow umbilicus in hasselquisti Ehrenberg, 1831 = Helix ehrenbergi Roth, 1839) E. d. desertorum. The shells of E. d. desertorum are often and E. kobelti (Westerlund, 1889) as separate species rib-striated, whereas those of E. desertorum irregularis are (Kaltenbach 1934, 1942; Heller 2009). smoother and only irregularly wrinkled. Some specimens

Fig. 1 Shell variability of Eremina desertorum in Egypt. —A. Eremina d. desertorum, Cairo (ZMH 45035). —B. D Eremina d. desertorum, El Alamein 100 km toward Cairo (ZMH 79772). —C. Eremina d. desertorum, El Alamein 65 km toward Cairo (ZMH 79776). —D. Eremina desertorum irregularis, Borg El Arab (ZMH 79768). —E. Eremina desertorum irregularis, Ezbet El-Sheik Sabir area S of El Hammam (ZMH 79769). Scale bar: E 10 mm.

ª 2015 Royal Swedish Academy of Sciences, 45, 1, January 2016, pp 48–61 49 Differentiation in Eremina desertorum in Egypt R. F. Ali et al. of E. desertorum irregularis have a more or less wide, Cairo, because this area is especially interesting for the wrinkled accretion at the peristome (Fig. 2B–C), which interpretation of the relationship of these two taxa. does not occur in E. d. desertorum. Differences in the size Kaltenbach (1934) mentioned a wide hybrid zone between of the radula, the number of radular teeth and the number E. d. desertorum and E. desertorum irregularis with a high of ridges on the jaw (Kaltenbach 1934) are apparently percentage of specimens of intermediate shell size between correlated with the body size. Hesse (1915) has not found 110 and 190 km north-west of Cairo. In contrast, anatomical differences between E. d. desertorum from Cairo Kaltenbach (1942) reported an abrupt transition between and E. desertorum irregularis from Ar Raml in Alexandria. E. desertorum irregularis and E. d. desertorum about 20– Eremina d. desertorum occurs in the interior of northern 30 km south of the Mediterranean coast, where he found a Egypt, approximately from Alexandria southwards to form of E. desertorum that he described as E. desertorum Fayyum and eastwards to the Gaza strip and Israel, whereas mariuti. Therefore, we sampled Eremina populations along E. desertorum irregularis is distributed along the Mediter- the Mediterranean coast region of Egypt and along a ranean coast from southern Tunisia through Libya transect from the Mediterranean coast toward Cairo. We eastwards to Alexandria and occurs also south-east of analysed variables of the shell and the genitalia with statis- Cairo. We focus on the region between Alexandria and tical methods that allow the separation of size and shape

A BC

F Fig. 2 Shell variability of Eremina desertorum irregularis in Egypt. —A. Specimen without peristomal accretion, El Dabaa (ZMH 79759). —B. Specimen with E H peristomal accretion, El Dabaa (ZMH 79759). —C. Specimen with wide peristomal accretion, El Sallum 183 km toward Mersa Matruh (ZMH 79767). —D–E. El Dabaa, recent (ZMH 79759). —F. El Dabaa, (sub-)fossil (ZMH 79872). —G–I. Mersa Matruh 180 km toward El G I Alamein (close to El Alamein) (ZMH 79764). Scale bar: 10 mm.

50 ª 2015 Royal Swedish Academy of Sciences, 45, 1, January 2016, pp 48–61 R. F. Ali et al. Differentiation in Eremina desertorum in Egypt components. Furthermore, we analysed the variation of biologically meaningful climatic variables (BIOCLIM data mitochondrial DNA in this region to characterize the two set; Nix 1986) were acquired from the WorldClim database taxa and their relationships and to investigate the causes of (http://www.worldclim.org; ca. 1 km2 resolution; Hijmans the morphological and genetic variation. et al. 2005). Precipitation of the driest month is 0 mm for all stations. Thus, this variable was not considered. Because Material and methods some of the other variables are highly correlated, we Study area and sampling converted them into a set of linearly uncorrelated variables We sampled Eremina populations along the Mediterranean using principal component analysis with varimax rotation as coast region of Egypt and along a transect from the implemented in the program SPSS Statistics version 21 Mediterranean coast toward Cairo (Fig. 3; see Supporting (IBM, Armonk, NY, USA). We investigated the inﬂuence information, Table S1). Specimens were killed in boiling of the environmental principal components on shell and water and preserved in 70% ethanol. Voucher specimens genital variables using stepwise multiple linear regression are kept in the Zoological Museum of the University Ham- analyses with SPSS. burg in Germany (ZMH). Additionally, vouchers to Kal- tenbach (1934, 1942) kept in the Senckenberg-Museum in Shell variables Frankfurt am Main, Germany (SMF), were used for shell Shells of 438 individuals from 12 populations of E. d. deser- measurements (see Supporting information, Table S1). torum and 18 populations of E. desertorum irregularis were There is sporadic rainfall from October to March along analysed (see Supporting information, Table S2). We the Mediterranean coast of Egypt. Precipitation is decreas- measured the shell diameter, diameter of the spire, shell ing toward the interior. Whereas the average annual height, diameter of the aperture and height of the aperture rainfall along the Mediterranean coast may reach c. (Fig. 4) with an electronic calliper and counted the number 150 mm, there are only about 20 mm of precipitation in of whorls with an exactness of 0.25 whorls following the Cairo and 8 mm south of Fayyum (Fig. 3). Several other method described by Kerney & Cameron (1979: 13). climatic parameters change geographically in a similar way as temperature. For example, annual mean temperature Genital variables varies between 18.8 and 19.6°C along the Mediterranean Genitalia of 141 individuals from six populations of coast and increases toward the interior up to 21.3°Cin E. d. desertorum and 14 populations of E. desertorum irregu- the vicinity of Fayyum. For each sampling locality, 19 laris were analysed (see Supporting information, Table S3).

25°E 30°E 33°E 32°N Alexandria

5 1 2 3 4 7 8 16 17 9 10 18 11 12 13 14 25 15 20 19 21 Cairo 22 6 23 24 26

27 28 29 Re 29°N d Sea 30

Nile

Fig. 3 Sampling localities (see Supporting information, Table S1) of Eremina desertorum irregularis (locality 1–18) and Eremina d. desertorum (locality 19–30) in northern Egypt. Shading: annual precipitation (20 mm zones, white: 0–20 mm). Histograms: percentage distribution of shell diameter in the sampled populations (5 mm intervals from 15 to 40 mm). The dashed line indicates the Nile delta.

ª 2015 Royal Swedish Academy of Sciences, 45, 1, January 2016, pp 48–61 51 Differentiation in Eremina desertorum in Egypt R. F. Ali et al.

da D Fig. 4 Shell of Eremina desertorum showing the taken measurements. D: shell diameter; da: diameter of the aperture; H: shell height; ha: height of the aperture; S: diameter of the spire.

We measured the length of the penis plus the distal O epiphallus, which could not be delimited without opening, Pd the proximal epiphallus (proximal and distal refer to the E DS position in relation to the gonad), the vagina distal of pE dV the insertion of glandulae mucosae, the vagina proximal of the insertion of the glandulae mucosae, the dart sac and F pV the oviduct with an ocular micrometre and noted the Fig. 5 presence or absence of a rudimentary flagellum (Fig. 5). Genitalia of Eremina desertorum showing the taken measurements. DS: dart sac; dV: vagina distal of the insertion of the glandulae mucosae; F: flagellum; O: oviduct; PdE: penis and Morphometric analyses epiphallus distal of the insertion of the penial retractor; pE: The results of ordinary principal component analysis (PCA) epiphallus proximal of the insertion of the penial retractor; pV: and linear discriminant analysis (LDA) cannot be easily vagina proximal of the insertion of the glandulae mucosae. translated into differential characteristics of taxa. Therefore, we applied multivariate ratio analysis (MRA; Baur & Leuen- DNA extraction, amplification and sequencing berger 2011) that allows the interpretation of the results Total genomic DNA was extracted from tissue samples of from PCA and LDA in terms of body ratios that can the foot preserved in 100% isopropanol following a slightly directly be used for classification. Moreover, MRA allows modified version of the protocol described by Sokolov distinguishing between size and shape components. We (2000) as detailed in Scheel & Hausdorf (2012). performed a PCA in MRA shape space (as defined by Baur Fragments of 16S rDNA were amplified by polymerase & Leuenberger 2011). The principal components (PCs) of chain reaction (PCR) using the primer pair 16Scs1 and shape are most strongly determined by ratios between vari- 16Scs2 (Chiba 1999; Sigma-Aldrich, St. Louis, USA). ables lying close to the opposite ends of the PCA ratio spec- Amplifications were performed in 25 lL volumes tra. The ratios between variables lying close to the opposite containing 2 lL109 amplification buffer B (biolabprod- l ends of the allometry ratio spectrum show the greatest ucts, Bebensee, Germany), 4 L MgCl2 (25 mM, amount of allometry. The LDA ratio extractor was applied biolabproducts), 1 lL dNTP mix (5 mM each, biolabprod- to find the body ratios that best discriminate the indicated ucts), 1 lL of each primer (10 lM), 0.2 lL Crystal Taq taxa. The measure d indicates how well size discriminates in DNA polymerase (biolabproducts), 1 lL template DNA l comparison with a ratio. The calculations were performed and 14.8 L ddH2O under the following reaction condi- with the software R (R Core Team 2012) and the R scripts tions: an initial denaturation step at 94°C for 2 min, 35 provided by Baur & Leuenberger (2011). Further discrimi- PCR cycles (94°C for 40 s, primer specific50°C for 40 s, nant analyses were performed with SPSS Statistics. 72°C for 40 s) and a final extension step at 72°C for

52 ª 2015 Royal Swedish Academy of Sciences, 45, 1, January 2016, pp 48–61 R. F. Ali et al. Differentiation in Eremina desertorum in Egypt

5 min. Prior to sequencing, PCR products were with MEGA) can be ascribed to the geographic enzymatically cleaned up by adding 0.65 lL thermosensi- relationships between the populations (latitude and tive alkaline phosphatase (Thermo Fisher Scientific, longitude were used as spatial variables) and in how far Waltham, MA, USA) and 0.35 lL exonuclease I (Thermo they are affected by environmental parameters. We used Fisher Scientific) to a 5 lL aliquot of the PCR followed by 9999 permutations to estimate P values. an incubation step at 37°C for 15 min. The enzymes were inactivated at 85°C for 15 min. Both strands of the Results amplified products were Sanger-sequenced at Macrogen Shell variation Europe Laboratory (Amsterdam, the Netherlands). The plot of the first shape PC against isometric shell size (defined as the geometric mean of all shell measurements, Alignment and analysis of DNA sequences see Baur & Leuenberger 2011) showed that there is little Forward and reverse sequences were assembled using overlap between E. d. desertorum and E. desertorum irregu- ChromasPro version 1.7.4 (Technelysium, Tewantin, laris, as classified by the criteria of Kaltenbach (1934) Australia). The sequences were aligned with MUSCLE concerning isometric size and that there was allometry (Edgar 2004) as implemented in MEGA 6.0.6 (Tamura indicated by the correlation of the shape component with et al. 2013) with the default settings. The 16S rDNA isometric size (Pearson correlation, two-sided, r = 0.789, sequences analysed in this study have been deposited in P < 0.000; Fig. 6A). Based on isometric shell size and the GenBank under accession numbers KP230672-KP230700. first shell shape component, 98.6% of the individuals were We constructed a median-joining network (Bandelt et al. correctly classified in a discriminant analysis (a priori, 1999) based on the 16S rDNA sequences using the program E. desertorum ‘mariuti’ was classified as E. d. desertorum). PopART (Leigh & Bryant 2014) with e = 0. We tested pre- The population described as E. desertorum mariuti by defined phylogenetic hypotheses in a maximum likelihood Kaltenbach (1942) was intermediate between the two taxa, framework. Maximum likelihood analyses were conducted but concerning size it agreed better with E. d. desertorum. with Treefinder, version of March 2011 (Jobb et al. 2004; Eremina d. desertorum and E. desertorum irregularis are also Jobb 2011). As appropriate model for sequence evolution, J2 more or less separated in shape space (Fig. 6B). Again, considering among-site rate variation with a five-category E. desertorum ‘mariuti’ is intermediate, but concerning discrete gamma-distribution for rates was determined with shape, it agreed better with E. desertorum irregularis. The the ‘propose model’ option of Treefinder based on the first shape PC explained 48.4% of the variance and was Akaike information criterion with a correction term for small mainly correlated with ratios like S/ha as indicated by the sample size. We used a constrained tree and the ‘resolve position of these variables at the opposite ends of the PCA multifurcations’ option of Treefinder to obtain the maxi- ratio spectrum (Fig. 6C). The second shape PC explained mum likelihood tree for a specified hypothesis. Then, we 39.3% of the variance and was mainly correlated with the investigated whether the maximum likelihood tree for this ratio S/da (Fig. 6D). The allometry ratio spectrum hypothesis is part of the confidence set of trees applying the indicated that ha and da (at the opposite ends of the spec- approximately unbiased test (Shimodaira 2002). trum) showed the greatest amount of allometry (Fig. 6E). To infer population size changes from the haplotype The LDA ratio extractor indicated that S/H was the most 0 data, we used Fu s Fs statistic (Fu 1997). Negative discriminating ratio between E. d. desertorum and E. deserto- departures of this statistic from zero indicate an excess of rum irregularis. The measure d, which indicates how well low-frequency alleles expected in the course of a popula- shape discriminates in relation to size (d close to one means fi tion expansion (Fu 1997). Con dence intervals of Fs were that separation is mainly due to size, whereas shape is impor- determined by coalescent simulations (1000 replicates). tant for a value close to zero), was 0.53 for the first ratio and, The calculations were performed with DnaSP version thus, the separation was about equally supported by size and 5.10.01 (Librado & Rozas 2009). shape. Most E. d. desertorum have S/H ratios >0.75, whereas most E. desertorum irregularis have S/H ratios <0.70. The Influence of geographic distance and environmental next best discriminating shell ratio as little correlated as pos- parameters on morphometric and genetic variables sible with S/H was da/ha. The standard distance of the best We used distance-based redundancy analysis (dbRDA; discriminating ratio that indicates its discriminating power Legendre & Anderson 1999; McArdle & Anderson 2001) (see Baur & Leuenberger 2011) was 3.1, and the standard as implemented in DISTLM (Anderson 2004) to test in distance of the second best was 2.0. While it is evident from how far Euclidean distances between the morphometric a plot of these ratios that none of the ratios alone is sufficient variables of the measured populations and genetic distances for discriminating between E. d. desertorum and E. deserto- between the 16S rDNA haplotypes (p-distances calculated rum irregularis (Fig. 7A), 92.7% of the individuals were

ª 2015 Royal Swedish Academy of Sciences, 45, 1, January 2016, pp 48–61 53 Differentiation in Eremina desertorum in Egypt R. F. Ali et al.

A B

0.2

0.1

0.0

–0.1

Shape PC1 (48.4%)

Shape PC2 (39.3%)

–0.2 –0.1

E. d. desertorum E. d. desertorum E. desertorum irregularis –0.3 E. desertorum irregularis

–0.2 E. desertorum'' mariuti E. desertorum'' mariuti –0.4 –0.2 0.0 0.2 0.4 –0.2 –0.1 0.0 0.1 0.2 Isometric size Shape PC1 (48.4%)

CDE

0.43 –0.58 0.64 –0.55 0.54 0.36 ha H D Sda D ha D H S H ha da Sda Fig. 6 Multivariate ratio analysis of shell measurements of Eremina d. desertorum and Eremina desertorum irregularis. —A. Plot of isometric size vs. first principal component in shape space. —B. Plot of first vs. second principal component in shape space. —C. PCA ratio spectrum of the first principal component. —D. PCA ratio spectrum of the second principal component in shape space. —E. Allometry ratio spectrum. Vertical bars in C-E = 68% confidence intervals based on 500 bootstrap replicates.

A B E. d. desertorum

2.5 E. desertorum irregularis

PdE/O

da/ha

01.52.0

1. E. d. desertorum

0.7 0.8 0.9 1.0 1.1 E. desertorum irregularis E. desertorum'' mariuti

0.5 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 2345 S/H PdE/dV Fig. 7 Plots of ratios best discriminating between Eremina d. desertorum and Eremina desertorum irregularis. —A. Shell ratios: S/H vs. da/ha. —B. Genital ratios: PdE/dV vs. PdE/O. correctly classiﬁed in a discriminant analysis based on both A plot of the most discriminating ratio, S/H, against ratios (96.9%, if the E. desertorum ‘mariuti’ individuals were longitude (Fig. 8A) showed an abrupt transition between not considered). E. d. desertorum and E. desertorum irregularis south of the

54 ª 2015 Royal Swedish Academy of Sciences, 45, 1, January 2016, pp 48–61 R. F. Ali et al. Differentiation in Eremina desertorum in Egypt

A B E. d. desertorum E. d. desertorum E. desertorum irregularis E. desertorum irregularis E. desertorum'' mariuti

0.85 0.90

0.80

345

S/H

PdE/dV

0.55 0.60 0.65 0.70 0.75 26 27 28 29 30 31 26 27 28 29 30 Longitude Longitude Fig. 8 Plots of ratios best discriminating between Eremina d. desertorum and Eremina desertorum irregularis against longitude. —A. Shell ratio S/H. —B. Genital ratio PdE/dV.

Mediterranean coast. As already noted, the intermediate best 0.9. Both are distinctly smaller than those of the two population, E. desertorum ‘mariuti’, agreed concerning best discriminating shell ratios. In accordance with that, shape with E. desertorum irregularis. only 72.3% of the individuals were correctly classified in a discriminant analysis based on these ratios (Fig. 7B). Genital variation A plot of the most discriminating ratio of genital organs, A rudimentary flagellum was found in 16.1% of the stud- PdE/dV, against longitude (Fig. 8B) showed no changes ied E. d. desertorum individuals and in 5.5% of the studied across the transition zone between E. d. desertorum and E. desertorum irregularis individuals. The plots of isometric E. desertorum irregularis south of the Mediterranean coast. size against the first shape PC derived from the genital measurements (Fig. 9A) and of the two-first shape PCs Principal component analysis of environmental variables (Fig. 9B) showed that neither size nor the ratios of the The principal component analysis of the values of 18 different parts of the genitalia (‘shape’) are sufficient to climatic variables (BIOCLIM data set) at the sampling separate E. d. desertorum and E. desertorum irregularis. Based localities extracted three meaningful (eigenvalues > 1) on isometric genital size and the first genital ‘shape’ principal components (PCs) accounting for 91.9% of the component, only 82.3% of the individuals could correctly total variation. PC1, accounting for 67.5% of the total vari- be classified in a discriminant analysis. The first shape PC ation, was a climatic gradient from cool and wet to warm explained 32.8% of the variance and was mainly correlated and dry sites. PC2 (14.7%) ordered sites mainly according with the ratio O/pV as indicated by the position of these to mean temperature of coldest quarter. PC3 (9.7%) is variables at the opposite ends of the PCA ratio spectrum most strongly correlated with precipitation of driest quarter (Fig. 9C). The second shape PC explained 25.8% of the and precipitation of warmest quarter. variance and was mainly correlated with the ratio pV/dV (Fig. 9D). The allometry ratio spectrum indicated that O Correlation of shell parameters with environmental and dV (at the opposite ends of the spectrum) showed the variables greatest amount of allometry (Fig. 9E). Climatic principal components 1 and 3 were included in The LDA ratio extractor indicated that PdE/dV was the the best model revealed by stepwise multiple regression most discriminating ratio. A value of d = 0.47 was (R2 = 0.499, P < 0.001) with shell isometric size as computed for the first ratio vector, and thus, the separation dependent variable. However, shell isometric size varied was about equally supported by size and shape. The next also with geography and the geographical positions of the best discriminating shell ratio as little correlated as possible sampled populations alone explained 50.6% of the observed with PdE/dV was PdE/O. The standard distance of the variation in shell isometric size (P < 0.001). Climatic PC1 best discriminating genital ratio was 1.4 that of the second still explained 6.9% of the variation in shell isometric size

ª 2015 Royal Swedish Academy of Sciences, 45, 1, January 2016, pp 48–61 55 Differentiation in Eremina desertorum in Egypt R. F. Ali et al.

A B E. d. desertorum E. d. desertorum

1.0 E. desertorum irregularis E. desertorum irregularis

0.5

0.0 0.2 0

0.0

Shape PC1 (32.8%)

Shape PC2 (25.8%)

–0.4 –0.2

–0.5

–0.6 –0.8 –0.6 –0.4 –0.2 0.0 0.2 0.4 –0.5 0.0 0.5 1.0 Isometric size Shape PC1 (32.8%)

C D E

0.49 –0.57 0.35 –0.52 0.50 0.30

pE PdE pE PdE O pV DS DS dV O pE PdE pV dV DS dV pV O Fig. 9 Multivariate ratio analysis of genital measurements of Eremina d. desertorum and Eremina desertorum irregularis. —A. Plot of isometric size vs. first principal component in ‘shape’ space. —B. Plot of first vs. second principal component in ‘shape’ space. —C. PCA ratio spectrum of the first principal component. —D. PCA ratio spectrum of the second principal component in ‘shape’ space. —E. Allometry ratio spectrum. Vertical bars in C-E = 68% confidence intervals based on 500 bootstrap replicates. if geographical coordinates were included as covariables P = 0.002). This may indicate a change of the regional cli- (P = 0.047). Climatic PC3 had no significant influence on mate. shell isometric size if geographical coordinates and climatic Climatic principal components 1 and 3 were included in PC1 were considered as covariables (P = 0.332). the best model revealed by stepwise multiple regression A plot of shell diameter vs. climatic PC1 showed that (R2 = 0.655, P < 0.001) with the first principal shell shape both E. d. desertorum and E. desertorum irregularis tended to component as dependent variable. The geographical become smaller with decreasing precipitation and increas- positions of the sampled populations alone explained ing temperature, but that E. desertorum irregularis remained 65.2% of the observed variation in the first principal shell larger than E. d. desertorum in hot areas in the interior with shape component (P < 0.001). Climatic PC1 explained little precipitation (Fig. 10, see also Fig. 3). Furthermore, 8.1% of the variation of the first principal shell shape the plot showed again, that the intermediate population, component if geographical coordinates were included as E. desertorum ‘mariuti’ better agreed with E. d. desertorum covariables (P = 0.008). Climatic PC3 explained still 3.9% concerning shell size. of the variation of the first principal shell shape component There is a sample of (sub-)fossil E. desertorum irregularis if geographical coordinates and climatic PC1 were included shells of unknown age from El Dabaa that are significantly as covariables (P = 0.045). larger than recent shells from the same locality (D = 33.4– Shell isosize explained 78.8% of the variation of the first 42.3 mm, mean 37.4 3.3 mm (n = 8) in the fossil ones principal shell shape component (P < 0.001). We tested the (Fig. 2F) vs. D = 28.7–38.3 mm, mean 31.9 2.1 mm (n hypothesis that the differences in shell shape were only a = 35) in the recent ones (Fig. 2A, D-E), t-test, two-sided, non-adaptive, allometric consequence from changes in shell

56 ª 2015 Royal Swedish Academy of Sciences, 45, 1, January 2016, pp 48–61 R. F. Ali et al. Differentiation in Eremina desertorum in Egypt size. This hypothesis could be rejected, because climatic measured populations were not significantly correlated with PC1 still explained a significant (P = 0.034), though small the climatic principal components. The geographical posi- part (3.4%) of the variation of the first principal shell shape tions of the sampled populations alone explained 35.6% of component if shell isosize was used as a covariable. the observed variation in genital isometric size (P = 0.031). There was no significant correlation of the first principal Correlation of genital parameters with environmental genital shape component with geography (P = 0.322). variables The variation in genital isometric size and the variation in Mitochondrial DNA sequences the first principal genital shape component of the 20 We sequenced a fragment of the mitochondrial 16S rDNA from 12 individuals from six populations of E. d. desertorum and 17 individuals from nine populations of E. desertorum E. d. desertorum irregularis. A total of 57 sites of the 818 sites without E. desertorum irregularis alignment gaps were polymorphic. Genetic p-distances E. desertorum'' mariuti were very small: 0.1–1.5%, mean 0.8 0.4% within E. d. desertorum; 0.0–1.5%, mean 0.8 0.4% within E. desertorum irregularis; and 0.2–1.7%, mean 0.9 0.3% between these taxa. The median-joining network showed

30 35 40 that the two taxa are not reciprocally monophyletic (Fig. 11). This was corroborated by an approximately D (mm) unbiased test (P < 0.001). Total nucleotide diversity p was ﬁ 0 0.00827. A highly signi cantly negative Fu s Fs statistic

25 (16.737, P < 0.001) indicated population expansion. A dbRDA based on the p-distances between the mitochondrial 16S rDNA sequences of all sampled individ-

20 uals revealed that isolation by distance explained 13.9% of the observed variation (P = 0.017). The correlation –1.0 –0.5 0.0 0.5 1.0 1.5 2.0 2.5 between the genetic distances and the climatic PC1 was Climatic PC1 marginally non-signiﬁcant (P = 0.051), but became non- Fig. 10 Plots of shell diameter of Eremina d. desertorum and signiﬁcant if geographical coordinates were included as Eremina desertorum irregularis against climatic PC1. covariables (P = 0.175).

3 22

18 12 11 11

3+12 15+16 25 10 23

10 15 23 24 21 21 24 25

1 individual 20 Fig. 11 Median-joining network based on 22 2 individuals partial 16S rDNA sequences of 29 20 Eremina desertorum individuals. Numbers 6 E. d. desertorum refer to populations (see Fig. 3 and E. d. irregularis Supporting information, Table S1). 6

ª 2015 Royal Swedish Academy of Sciences, 45, 1, January 2016, pp 48–61 57 Differentiation in Eremina desertorum in Egypt R. F. Ali et al.

Discussion population. Unfortunately, no new material could be Pattern of differentiation collected from this population. The morphometric analyses showed that E. d. desertorum In contrast to the sharp boundary seen in the shell and E. desertorum irregularis are differentiated with regard variables, there is no clear separation of E. d. desertorum to shell size as well as to shell shape (Figs 6A–B, 7A, 8A and E. desertorum irregularis with regard to genital and 10), although there is some overlap in the variation of measurements (Figs 7B, 8B and 9). Even closely related these two taxa. The plot of the most discriminating ratio, helicoid land snail species differ often in ratios of the S/H, against longitude showed a sharp boundary between genital organs indicating reproductive isolation (e.g. Subai the E. d. desertorum and E. desertorum irregularis (Fig. 8A) 2005; Neubert 2014). Because of the lack of differences in indicating that there is either little gene flow across the the genitalia, the lack of reciprocal monophyly of the mito- boundary or that there is strong selection against interme- chondrial haplotypes of E. d. desertorum and E. desertorum diate specimens. The notes of Kaltenbach (1934, 1942) on irregularis (Fig. 11), and the existence of transitional speci- the changes of the shell variability of Eremina between mens and a transitional population with regard to shell Cairo and Alexandria were contradictory. Kaltenbach characters, we prefer to classify these two taxa as subspecies (1934) mentioned a wide hybrid zone between E. d. deserto- in accordance with Biggs (1959). rum and E. desertorum irregularis with a high percentage of specimens of intermediate shell size between 110 and Process of differentiation 190 km from Cairo. Actually, the variability of shell size of Given the high estimates of the divergence rate of 16S E. d. desertorum increases from the boundary with E. deser- rDNA in helicoids varying between 3.2 and 11.2% per torum irregularis toward Cairo. However, the differentiation million years (e.g. Chiba 1999; Hayashi & Chiba 2000; in shell shape is not affected (Fig. 8A). Thus, there is no Watanabe & Chiba 2001; Van Riel et al. 2005), the low evidence that the increase in size variability is the result of genetic distances between the 16S rDNA haplotypes of introgression from E. desertorum irregularis. Our results are E. desertorum (at most 1.7%) indicate that the differentia- in better agreement with Kaltenbach’s (1942) point of view tion between E. d. desertorum and E. desertorum irregularis who considered the populations along the desert road from started at most c. half a million years ago, perhaps it is Cairo to Alexandria to be typical E. d. desertorum. His note actually only 150 000 years old. There are no major that the shells along the road are sometimes larger and geographic barriers (mountains, rivers, etc.) between the sometimes smaller does not describe the pattern correctly. ranges of these taxa. Thus, the differentiation might have A plot of shell diameter against a climatic gradient from started, while the populations were in contact. However, it cooler and more humid conditions along the Mediter- is possible that the ranges of the two snail taxa were sepa- ranean coast of Egypt to arid and hot conditions in the rated by a westward extension of the Nile delta (see Fig. 3) interior (Fig. 10) showed that shell size does not vary during more humid periods. Habitats in the Nile delta are randomly along this gradient, but that average shell diame- unsuitable for these desert snails because they are too ter tends to increase with increasing precipitation and humid. Such habitats might have formed a barrier between decreasing temperature toward the coast. However, the the E. desertorum irregularis populations along the populations close to the boundary with E. desertorum Mediterranean coast in the west and the E. d. desertorum irregularis show a slightly smaller average shell diameter populations in the inland in the south-east. There were than those further south-eastwards. This contributes to the several humid intervals in the region during the period in abrupt transition between the two taxa, which Kaltenbach which E. desertorum differentiated (Vaks et al. 2006). After (1942) also noted. The average shell diameter of the the regression of the supposed extension of the Nile delta neighbouring E. desertorum irregularis is also slightly in a more arid period, both subspecies came into contact 0 smaller than that of E. desertorum irregularis populations west of the Nile delta. Actually, Fu s Fs statistic of the from other areas. There is only a narrow hybrid zone mitochondrial sequence data is significantly negative between the ranges of E. d. desertorum and E. desertorum indicating a population expansion in agreement with other irregularis c. 20 km south of the Mediterranean coast west evidence that the Nile region in northern Egypt was more of Alexandria, where Kaltenbach (1942) sampled an humid well into historical times than today and that desert intermediate population that he named E. desertorum expanded, probably as a result of vegetation changes caused mariuti. This population agrees better with E. d. desertorum by human action (Reale & Dirmeyer 2000; Reale & Shukla with regard to shell size (Fig. 10), but corresponds with 2000). The finding of (sub-)fossil shells of E. desertorum E. desertorum irregularis concerning the lower S/H ratio irregularis near El Dabaa that are significantly larger than (Fig. 8A). Thus, we consider this population as a hybrid recent ones from the same locality (Figs 2F vs. 2A, D-E) is

58 ª 2015 Royal Swedish Academy of Sciences, 45, 1, January 2016, pp 48–61 R. F. Ali et al. Differentiation in Eremina desertorum in Egypt also in accordance with a decrease of precipitation in that from changes in shell size, because climatic variables area. explained still a significant, although small part of the vari- Nevertheless, the differences between E. d. desertorum ation of the first principal shell shape component if shell and E. desertorum irregularis cannot simply be ascribed to isosize is used as a covariable. neutral historical changes in allopatry. Non-adaptive clines A more arid climate not only selects for smaller body produced by genetic drift while populations were separated size by shorter feeding periods, but also for a smaller shell are rapidly eroded with increasing gene flow (Endler 1973). aperture to reduce water loss. Eremina d. desertorum, which The sharp border between E. d. desertorum and E. deserto- occupies the more arid part of the study region, responded rum irregularis concerning shell shape and size (Figs 8A to the selection for smaller body size by a less rapidly and 10) that are at least partly encoded by nuclear genes is increasing body whorl in comparison with E. desertorum in contrast with the lack of a separation with regard to the irregularis (Figs. 1–2) as indicated by the higher S/H ratio supposedly neutral mitochondrial marker (Fig. 11). Given than in E. desertorum irregularis (Figs 7A and 8A). A less that the effective population size of nuclear genes is rapidly increasing body whorl entails a disproportionally fourfold as high as that of mitochondrial markers, we smaller diameter of the aperture with regard to the diame- would expect that differences in mitochondrial markers ter of the shell. Furthermore, the area of the aperture is would be fixed much faster than differences in nuclear reduced in E. d. desertorum by a disproportional reduction genes by neutral processes. Thus, selection is probably of its height relative to its diameter as indicated by the involved in the differentiation in shell shape and size. allometry ratio spectrum (Fig. 6E) and the higher da/ha Actually, we found a significant correlation of shell size ratio than in E. desertorum irregularis (Fig. 7A). In agree- and shape in E. desertorum and a climatic gradient from ment with the results of the dbRDAs, these considerations cooler and more humid conditions along the Mediter- suggest the shape differences between E. d. desertorum and ranean coast of Egypt to arid and hot conditions in the E. desertorum irregularis are partly an indirect consequence interior (Fig. 10). Shell size of E. desertorum increases with of selection for body size, but are also directly affected by increasing humidity. A relationship between shell size and selection for reduction of aperture size. precipitation has also been found in other land snail species In addition, shell shape might be affected by the differ- in other dry regions where humidity is a limiting factor ent behaviour and the preferred habitats of E. d. desertorum (Sacchi 1965; Heller 1979; Magaritz & Heller 1980; Gould and E. desertorum irregularis. Eremina d. desertorum occurs 1984; Goodfriend 1986). These results are in conflict with mainly in sandy deserts and feeds at shrubs and rests on the expectations based on the hypothesis that large snails these shrubs, under stones or in rock crevices, whereas are superior in dry habitats, because they lose relatively less E. desertorum irregularis prefers stone deserts, where it feeds water because of their lower surface to volume ratio (Nevo mainly on lichens and rests attached to the sun-exposed et al. 1983). Obviously, the decisive selection pressure in side of stones (Kaltenbach 1934, 1942). The flatter shells the case of E. desertorum is not the increased relative water of E. d. desertorum may be an adaption to moving in dense loss of small individuals in dry environments. Rather, the shrubs and resting in narrow spaces under stones or in decrease of body size with decreasing precipitation might rocks. be explained by the limited time for activity and food intake in the more arid regions, whereas the snails may Acknowledgements grow larger in regions where sufficient humidity permits a We are grateful to R. Janssen for access to the collection longer period of ingestion (Goodfriend 1986). Larger vari- of the Senckenberg-Museum in Frankfurt am Main and to ants might have a selective advantage in regions with a the DAAD for funding the stay of RFA in Germany. higher precipitation because of a higher fecundity, but have a selective disadvantage in environments with less humidity References where there is not enough time for ingestion so that they Anderson, M. J. (2004). DISTML v.5: a FORTRAN Computer Pro- starve (Hausdorf 2006). gram to Calculate a Distance-Based Multivariate Analysis for a Lin- Gould (1984) supposed that even if one shell variable ear Model. Auckland: University of Auckland, Department of like shell size had an adaptive trigger, variations in other Statistics. € shell variables represent just non-adaptive, allometric Bandelt, H.-J., Forster, P. & Rohl, A. (1999). Median-joining net- fi consequences. Actually, shell shape varies with shell size works for inferring intraspeci c phylogenies. Molecular Biology and Evolution, 16,37–48. 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