Along a latitudinal gradient of ca. 7180 km of coastline—from the Pacific to the Atlantic Ocean, we showed evidence of a discontinuity in genetic and spermatic morphotypes at 37° S, with two distinct genetic lineages (clades) in males of
P. purpuratus. Although the presence of two lineages could be indicative of cryptic species for this intertidal mussel [
6,
21,
22], we interpreted our observation of a hybridization zone at 38° S as a strong signal of incipient or
in progress (
in fraganti) speciation processes occurring in
P. purpuratus [
6].
4.1. Sperm Morphology
In
Perumytilus purpuratus, there was evidence of geographical variability in sperm sizes (for the mean acrosome and head lengths). The AICc-selected model 2, an ANCOVA with Locality and Head as predictor variables of Acrosome length, was better than the others, including model 3 (ANOVA), showing that the north lineage was characterized by the shorter morphotype and the south lineage was characterized by localities with two different intermediate and long morphotypes, as shown in
Figure 2c,d.
Thus, our morphological findings revealed the following: (1) three sperm morphotypes along the Chilean and Argentinean distributional range of
P. purpuratus; (2) a main morphological break between northern and southern localities situated in the Pacific Ocean at 37° S; (3) along with an intermediate morphotype, and a novel morphotype, which was longer than those that were previously reported [
5,
6,
54] and was observed in southern localities of both the Pacific and Atlantic.
Several researchers have suggested that sperm traits are species-specific and, therefore, are valuable for studying taxonomic affinities among species (e.g., [
5,
6,
7,
55,
56]). In this same line, this detailed morpho-anatomical, ultrastructural, and molecular work in bivalves highlights that the ultrastructural characteristics of sperm are among the best morphological features for determining bivalve clades [
57]. Therefore, a relationship between sperm morphotypes and genetic structure was expected.
4.2. Linking Sperm Morphotypes with Genetic Divergence
In agreement with previous reports [
31,
32], we confirmed DUI for the mitochondrial genome of
P. purpuratus. In our preliminary assay, the 16S sequences from males showed a unique mitochondrial genome (M type) in the posterior adductor muscle, since no male sequences were observed in the female clade (see
Figure S2).
In molecular analyses of mussels, DNA is generally extracted from the posterior adductor muscle (or mantle) tissue. In
Brachidontes genera (a closely–related species), DUI has been found in some species, but not in others [
58,
59], and some researchers have opted for isolating DNA from the posterior adductor muscle to avoid using male gonadal tissue, which is enriched with paternally transmitted ‘‘male’’ mitochondrial genomes (i.e., M type) [
44,
45]. This was in accordance with our preliminary assay, since, in the female and male sequences of our outgroup species
Brachidontes rodriguezii, no gender-associated divergence was observed (see
Figure S2). Nevertheless, according to Trovant et al. (2013) [
45], the infiltration of the muscle tissue by the male germ line is unlikely; therefore, it is expected to be dominated by maternally transmitted mitochondria (F type), irrespective of the gender of the individual mussel sampled [
45]. However, the opposite was observed in males of
P. purpuratus, indicating that this should not be considered a rule within the Mytilidae family.
The molecular results obtained using mitochondrial 16S M-type and nuclear (28S) sequences showed genetic divergence and the presence of a phylogeographic structure along the distributional range of
P. purpuratus, with two well-differentiated north and south clades, which we have now designated as the north lineage and the south lineage. These results were congruent with the divergence observed in the 16S F type (i.e., according to our preliminary assay;
Figure S2) and with previous reports using mitochondrial (16S, COI) and nuclear (18S, 28S) molecular markers [
6,
21,
22,
46]. Our results also agreed with the regional genetic differentiation between
P. purpuratus from the southeastern Pacific (Punta de Tralca, 33°26′ S) and southwestern Atlantic (Puerto Lobos, 41°42′ S) found when using microsatellite markers [
60]. Overall, these findings are evidence for restricted gene flow between the north and south lineages.
In sessile marine broadcast spawners, such as
P. purpuratus, the connectivity level and genetic flow between distant populations can depend on multiple factors—for example, the larval dispersal capacity, contemporary oceanography, and historical climatic events (see [
22]). However, the relationship between genetic divergence and the acrosome morphotypes reported here, with the short morphotype associated with the north lineage and both intermediate and long morphotypes associated to the south lineage, suggests that reproductive barriers and, therefore, reproductive isolation could be the current mechanism preventing gene flow between lineages.
If we consider the species specificity of spermatozoan traits to differentiate closely related species (e.g., acrosome size as a species-conservative trait), then each morphotype must be a different species. However, our morpho-genetic results were contrasting because the two genetic lineages were associated with three sperm morphotypes.
On the one hand, the presence of two genetic lineages (the present work and [
6,
21,
22,
46]) suggests the evolutionary scenario of cryptic speciation [
6,
22,
54], which means two species with prezygotic isolation, i.e., barriers that prevent fertilization, and, therefore, genetic exchange between previously interbreeding populations [
61,
62]. However, the lack of genetic differentiation observed between the intermediate and long morphotypes—as both were grouped within the south lineage—may also suggest an evolutionary scenario of incipient species within the southern region. In reproductive terms, this scenario likely involves postzygotic isolation, which is characterized by barriers occurring after zygote formation, leading to the production of non-viable or infertile offspring [
61,
62].
4.3. Cryptic and Incipient Species Scenarios: An Evolutionary Perspective
According to our results, the scenarios of both cryptic and incipient species are probable. In this way, we hypothesize that the short morphotype represents a cryptic species and that individuals with intermediate and/or long morphotypes could correspond to incipient species.
In an evolutionary context, the association between genetic divergence and adjusted acrosome morphotypes that we observed could be explained by historical events, such as the LGM [
6,
21,
22], where glacial sheets most likely disrupted larval dispersal and, consequently, the genetic connectivity between the southern and northern populations. For example, glaciation–deglaciation events have shaped the geomorphological configuration of the Chilean coast. In this context, the coastline between Perú and Canal de Chacao (north of Chiloé island, ca. 41°47′ S) is continuous, smooth, and without breaks or major geographical features [
63]. However, from Chiloé to Cabo de Hornos (~56° S), the area known as the Chilean archipelago, the coastal geomorphology is complex and characterized by a profusion of gulfs, fjords, and channels resulting from the combined effects of tectonic processes and glaciation [
63] (
Figure 1). During the Last Glacial Maximum (LGM), which is dated 23–25 ka ago for Patagonia [
64,
65], icefields covered all of southern Chile, from the Chilean Lake District (40° S) to Bahía Inútil in Tierra del Fuego (53.5° S) [
64], creating a great geographical barrier or breaks in habitat continuity between the southern and northern populations of
P. purpuratus.
In hypothetical terms, evidence has indicated that the north lineage corresponds to isolated populations that remained unaffected by icefields during the LGM period, and, therefore, larval connectivity and gene flow between the populations remained. This could explain the lower morphological variability in sperm observed for the north lineage, where the short morphotype remained relatively constant along ca. 1744 km of coastline (from Antofagasta to Lota;
Figure 2c). In the Antofagasta area (23° S), fossil records of
P. purpuratus observed in molluscan assemblages of the last interglacial period (early Pleistocene ca. 120 ka [
53,
54,
66,
67]) support this hypothesis. Consequently, our demographic analyses using mtDNA M-type showed a bimodal mismatch distribution for the north lineage (
Figure 7a). Dawson et al. (2002) [
68] suggested that a bimodal mismatch distribution is attributable to a historically differentiated allopatric population. In addition, bimodal shape may be sensitive to the age of the expansion, with the right peak representing an older expansion and the other peak a recent expansion [
69,
70]. In this way, the north lineage of
P. purpuratus might have a more complex evolutionary history than that of the south lineage. Instead, the mismatch distribution for the south lineage was unimodal in shape and closely fitted the expected distribution under the sudden expansion model (
Figure 7b), that may be attributable to a more recent population expansion than that of the north lineage. This finding was consistent with those of analyses conducted by Trovant et al. (2015) [
21] when using the COI molecular marker, as they (assuming a mutation rate of 0.19 substitutions/Myr) estimated different population expansion timings for each
P. purpuratus lineage, with a northern expansion that could have started around 15 ka (end of the Pleistocene) and a more recent expansion for the south lineage, where the largest change in population size could have occurred during the Holocene (11.5 to 3.5 ka BP). This could explain the variability in sperm morphotypes assigned to the south lineage, where the two morphotypes were distributed along ca. 5436 km of coastline, without a significative relationship with latitude (
Figure 2c).
Furthermore, our comprehensive sampling effort enabled us to precisely determine the latitudinal position of the morpho-genetic break, which was identified at 37° S on the Pacific coast. This location was notably farther north than the area covered during the Last Glacial Maximum (LGM). This evidence and the observation of the long morphotype in the break zone (Lebu) strongly suggest the postglacial recolonization of the south lineage of
P. purpuratus and support the hypothesis of ice-free refugia or suitable niches within these quaternary glacial areas [
71]. Therefore, we hypothesized that on the Pacific coast, the postglacial recolonization of the south lineage of
P. purpuratus reached 37° S and that the different sperm morphotypes of the south lineage could have originated in a distinct glacial refugium during the LGM period.
In other marine species inhabiting southern Chile, evidence of historical influences (i.e., the LGM) on cladogenesis also have been reported, such as in macroalgae [
72,
73,
74], fish [
75], arthropods [
76], and gastropods [
71,
77]. For example, in the red algae
Mazzaella laminarioides, Montecinos et al. (2012) [
72] also localized a genetic break at 37° S that, according to these authors, could have originated due to transient habitat discontinuities driven by episodic tectonic uplifting of the shoreline around the Arauco region (37°–38° S). In addition, three divergent lineages—northern (28°55′ S to 32°37′ S), central (34°05′ S and 37°38′ S), and southern (39°40′ S to 54°03′ S)—and evidence of postglacial recolonization from a northern glacial refugium area were observed in this species [
72]. In kelp, genetic disjunction between Patagonian (49°–56° S) and northern populations (32°–44°) was observed in
Durvillaea antarctica [
73]; in
Macrocystis pyrifera, Macaya and Zuccarello (2010) [
74] reported a genetic break at 42° S (Chiloé Island) and shared haplotypes among some of the subantarctic islands and southern-central Chile, suggesting a recent colonization of the subantarctic region. In
P. purpuratus, we observed shared haplotypes at the intra-lineage level, which was a signal of gene flow disruption between the north and south lineage. However, an exception was observed for IM Caleta Derrumbe, IM Punta Los Piures, and Tirúa (localities situated at 38° S; see
Figure 1), where some individuals showed the typical southern haplotypes, but others showed northern haplotypes with both mitochondrial (16S) and nuclear (28S) molecular markers. As a result, these individuals were positioned inside both the north and south lineage (see
Figure 4). We interpreted this outcome as evidence of a local hybridization zone at 38° S, suggesting the presence of an incipient speciation process within the south lineage.
4.4. Incipient Species and Postzygotic Isolation inside the South Lineage of P. purpuratus?
Our study allowed us to determine a possible hybridization zone at 38° S in the Pacific near the morpho-genetic boundary situated at 37° S. At 38° S, two principal localities were sampled: Tirúa (38°20′ S) in the continental territory and Isla Mocha (Punta Los Piures, Caleta Derrumbe, and Faro Viejo), situated 35 km in front of Tirúa in the insular territory. With exception of IM Faro Viejo, the individuals from these localities showed haplotypes that were typical of both the north and south lineage (see
Figure 4 and
Figure 5). It should be noted that we did not have sperm morphological information for Tirúa, but according to our findings, we hypothesized that the intermediate morphotype should be present in this locality. In this way, individuals at these localities showed the intermediate sperm morphotype, which was the morphotype attributed to the south lineage. However, we consider this finding as the first evidence of a secondary contact zone in
P. purpuratus, with individuals showing interspecific hybridization, where northern alleles probably introgressed into the gene pool of the south lineage.
In theory, a secondary contact zone is formed when two populations or lineages that have diverged due to genetic drift or selection during a period of geographic isolation come into contact [
78,
79,
80]. As was discussed previously, this finding was consistent with the hypothetical origin of divergence between the
P. purpuratus lineages and the later demographic expansion of the south lineage until 37° S. In this way, when secondary contact zones are established, genetic isolation can be maintained by prezygotic and/or postzygotic mechanisms [
80].
In the case of postzygotic isolation, the mechanisms for reproductive isolation have not been completed; therefore, hybrid offspring are produced through introgressive hybridization processes and maintained through intrinsic genetic incompatibilities or extrinsic causes of selection against hybrids [
80]. According to introgressive hybridization, the movement of alleles from one species into the gene pool of another divergent species occurs by means of repeated backcrossing of an interspecific hybrid with one of its parent species [
81,
82]. In mussels, hybridization zones have been well studied, especially in the
Mytilus species complex, which hybridizes naturally [
83,
84,
85,
86,
87], and the general expectation is that hybridization could lead to offspring with reduced fitness—for example, sterility or inferior viability—which is probably due to the assumption that crosses between divergent genotypes will always disrupt co-adapted genomes; however, fitness for hybrids can range to the highest to the lowest (see [
88]). In fact, several studies of
Mytilus have shown reduced hybrid fitness, such as through larval inviability and an early heterosis rate [
89], abnormal larvae [
90,
91], and high levels of sterility [
92]. In our work, the lowest frequency of “hybrid” specimens could be a signal of reduced fitness.
Regarding this, the genetical analyses performed using the GENELAND package, which is suitable for hybrid zone inference [
93], showed a transition cluster in IM Punta Los Piures and Tirúa (
Figure 5b), i.e., the localities in which individuals with both northern and southern haplotypes were observed. Nevertheless, the molecular markers used in this study (16S and 28S) are not suitable for the detection of hybrids. Therefore, whether individuals from these localities correspond to hybrids must be determined using specific markers for introgression and hybridization detection, such as SNPs, and this marker should be used to evaluate our hypothesis of a hybridization zone at 38° S.