Alopia

Koch, Eva L., Neiber, Marco T., Walther, Frank & Hausdorf, Bernhard, 2020, Patterns and processes in a non-adaptive radiation: Alopia (Gastropoda, Clausiliidae) in the Bucegi Mountains, Zoologica Scripta 49 (3), pp. 280-294 : 284-287

3 | RESULTS

3.1 | Mitochondrial gene tree and sequence diversity

Maximum-likelihood analyses of cox1 sequences (655 positions) of the Alopia taxa and one Albinaria puella puella and one Herilla ziegleri dacica as outgroups showed that the Alopia specimens from the Bucegi Mountains form three strongly supported clades, representing A. pomatias , A. straminicollis and the A. livida group including A. fussi and A. nixa ( Figure 1 ). Within A. pomatias p-distances reached 0.6%, within the A. straminicollis clade 5.3% and within the A. livida clade 4.2%; between the three clades p-distances reached 10.1% (Table S3).

The relationships between the three clades were not robustly resolved. Within the A. livida group, only the A. nixa specimens formed a well-supported monophylum, whereas individuals identified as A. livida nubila , A. livida bipalatalis , A. livida hypula and A. fussi intermingle. In the A. straminicollis clade, the individuals from the northern slope of the Bucegi Mountains representing A. s. straminicollis formed the sister group of the individuals from the southern part of the mountains representing A. straminicollis monacha . Among A. straminicollis monacha , there was also a dextral specimen from the hybrid zone with A. livida , identified as A. livida because of its dextral coiling. Likewise, there was a sinistral specimen from the hybrid zone between A. s. straminicollis and A. livida in the A. livida group that was identified as A. s. straminicollis because of its sinistral coiling.

3.2 | Network based on AFLP data

Using six primer combinations, we scored 2,215 AFLP fragments of 50–500 bp length in 250 Alopia specimens (Table S4). In contrast to the mitochondrial gene tree, the individuals belonging to one population usually form a cluster in the neighbour-net based on the nuclear AFLP markers ( Figure 2 ). The only species that is separated from the other species by a long branch is A. pomatias . The other taxa form an almost star-like radiation. The arrangement of the other populations in the network reflects their geographical relationships. At the one side of the network, the array starts with the A. straminicollis monacha populations from the southern part of the Bucegi Mountains (see Figure S1). Then, the hybrid population between A. straminicollis monacha and A. livida nubila (population 2) and the populations of A. livida nubila follow. The next branches are populations from higher altitudes classified as A. livida kimakowiczi and A. fussi . Among the A. fussi populations, the populations of A. nixa form a distinct cluster. Only one individual of A. nixa is separated from this cluster. The populations of A. livida bipalatalis , which are geographically placed between A. fussi and A. livida hypula , are also placed between these taxa in the network. Finally, the hybrid population between A. livida hypula and A. s. straminicollis (population 20) forms the transition to A. s. straminicollis .

3.3 | Population genetic structure

The ad hoc quantity Δ K, proposed by Evanno et al. (2005) to estimate the number of clusters, shows a maximum for K = 2 (Figure S2B). However, a plot of the likelihood of K for K = 1–12 showed that L (K) does not reach a plateau in this range, but that it further increases with higher values (Figure S2A). Thus, we show the STRUCTURE results for K = 2–8 that give additional insights into the genetic structuring of the Alopia populations in the Bucegi Mountains and the gene flow between population groups (Figure 3).

With K = 2, the two subspecies of A. straminicollis and A. livida hypula are separated from the other taxa of the A. livida complex. With K = 3, A. straminicollis monacha is separated from A. s. straminicollis % A. livida hypula . With K = 4, A. livida nubila and A. nixa % A. fussi are separated. With K = 5, A. pomatias albicostata , which is shown as an admixed population with lower K values, is separated as a distinct cluster. With K = 6, A. s. straminicollis and A. livida hypula are separated. With K = 7, populations 3 and 8 of A. livida nubila are separated. With K = 8, STRUCTURE runs show different groupings. In the run with the highest likelihood, A. nixa and A. fussi are separated and the division of the A. livida nubila populations disappears. Alternative classifications found in other runs show the former separation of populations 3 and 8 of A. livida nubila , but combine A. s. straminicollis and A. livida hypula in one cluster. In all results with K = 8, an additional cluster is found that never reaches a proportion above 75% and does not represent a specific taxon or population group. When K is further increased, the additional clusters do not represent distinct population groups.

The STRUCTURE analysis provides evidence for extensive gene flow between A. straminicollis and A. livida . In the contact zones (2 and 20), all individuals have a mixed ancestry and also in the neighbouring populations several individuals show high genetic proportions of the other species. There is also admixture between the other enantiomorph pair, A. nixa and A. fussi when they form separate clusters with K = 8 ( Figure 3 ). Individuals of populations 6 and 7 of A. nixa show approximately 30% genetic proportion of A. fussi and one individual of A. fussi has an inferred ancestry of 50% from the A. nixa cluster. There is also evidence for gene flow between A. livida and A. fussi (Figure 3). In population 11 of A. fussi some of the individuals show up to 50% inferred ancestry to A. livida nubila and the individuals of A. livida kimakowiczi have an inferred ancestry of 30% up to 50% from the A. nixa + A. fussi cluster. In contrast, there is no admixture between A. pomatias and A. nixa , despite they occur sympatrically.

BAPS recognized 6 clusters that correspond to A. pomatias albicostata , A. straminicollis monacha % A. livida nubila from the hybrid population (population 2), A. livida nubila (populations 3, 8) % 2 specimens of A. fussi from population 11, A. nixa % A. fussi % A. livida bipalatalis % A. livida kimakowiczi , A. livida hypula % A. s. straminicollis from the hybrid population (population 20) and A. s. straminicollis . The BAPS admixture solution showed less admixture than the STRUCTURE analysis with K = 6 (Figure 3).

An analysis of gene flow between predefined population groups corresponding to A. pomatias albicostata , A. straminicollis monacha , A. livida nubila , A. livida kimakowiczi , A. nixa , A. fussi , A. livida bipalatalis , A. livida hypula and A. s. straminicollis using a gene flow plot calculated with BAPS (Figure S3) indicated that gene flow downhill is distinctly higher than uphill in four of five cases in which gene flow exceeds 1% at least in one direction (Table S5). The exception is a higher amount of gene flow from A. livida nubila to A. fussi at higher altitudes.

An analysis of molecular variance (Table S6) attributed only a small part of the genetic variation (13%) to the division into five species proposed by Nordsieck (2008, 2007, 2016). The variation between populations accounted for 26% and the variation within populations for 61% of the total variation.

Figures

FIGURE 1 Maximum-likelihood tree based on partial cox1 sequences of 62 Alopia individuals from the Bucegi Mountains and Albinaria p. puella and Herilla ziegleri dacica as outgroups. Maximum-likelihood bootstrap values above 50 are indicated at the branches. Encircled numbers refer to sampling localities and colours refer to Alopia taxa (see Figure S1 and Table S1). Coiling direction is indicated by D = dextral or S = sinistral after the taxon names [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 2 Neighbour-net based on Jaccard distances between AFLP data of 250 Alopia individuals from the Bucegi Mountains. Encircled numbers refer to sampling localities and colours refer to Alopia taxa (see Figure S1 and Table S1). Coiling direction is indicated by D = dextral or S = sinistral after the taxon names [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 3 Results of the STRUCTURE (for cluster numbers from 2 to 8) and BAPS analyses of AFLP data of 250 Alopia individuals from the Bucegi Mountains. Coiling direction is indicated by D = dextral or S = sinistral after the taxon names [Colour figure can be viewed at wileyonlinelibrary.com]