Cophylogenetic analysis suggests cospeciation between the Scorpion Mycoplasma Clade symbionts and their hosts

Abstract

Scorpions are predator arachnids of ancient origin and worldwide distribution. Two scorpion species, Vaejovis smithi and Centruroides limpidus, were found to harbor two different Mollicutes phylotypes: a Scorpion Mycoplasma Clade (SMC) and Scorpion Group 1 (SG1). Here we investigated, using a targeted gene sequencing strategy, whether these Mollicutes were present in 23 scorpion morphospecies belonging to the Vaejovidae, Carboctonidae, Euscorpiidae, Diplocentridae, and Buthidae families. Our results revealed that SMC is found in a species-specific association with Vaejovidae and Buthidae, whereas SG1 is uniquely found in Vaejovidae. SMC and SG1 co-occur only in Vaejovis smithi where 43% of the individuals host both phylotypes. A phylogenetic analysis of Mollicutes 16S rRNA showed that SMC and SG1 constitute well-delineated phylotypes. Additionally, we found that SMC and scorpion phylogenies are significantly congruent, supporting the observation that a cospeciation process may have occurred. This study highlights the phylogenetic diversity of the scorpion associated Mollicutes through different species revealing a possible cospeciation pattern.

Fig 1. Sampling sites of scorpions in Mexico.

Map of Mexico showing the sampling locations, number of individuals and percentage of the different genera collected. Pie chart centres are positioned over the sampling locations. The size of the pie charts corresponds to the number of total individuals collected and colors represent the proportions of the different genera sampled.

Fig 2. Mollicutes 16S rRNA phylogenetic tree.

A phylogenetic tree was constructed with maximum likelihood and the GTR + G model using 237 sequences representing different species and the new lineages found in scorpions. Streptococcus pneumoniae, Bacillus subtilis and Bacillus cereus were used as outgroups. Bootstrap values above 60 are shown. Representative Mollicutes clades were collapsed (See S1 Fig for the non-collapsed tree).

Fig 3. Comparison of scorpion and symbiont phylogenies.

Different colors were used for each genus or clade: dark blue, Vaejovis; light blue, Mesomexovis; dark green, Thorellius; light green, Hoffmanihadrurus; purple, Megacormus; pink, Diplocentrus; red, Centruroides, “striped group”; yellow, Centruroides, “gracilis group”; brown, Androctonus; black, outgroups (Mastigoproctus sp. or Mycoplasma hyorhinis). (A) Scorpion phylogeny with concatenated 16S rRNA, CO1 and 28S rRNA genes (GTR + G + I). (B) SMC 16S rRNA gene phylogeny (GTR + G). (C) SG1 16S rRNA gene phylogeny (GTR + G).

Fig 4. Tanglegrams of host species and SMC phylogenies used for cophylogenetic analyses.

Scorpion phylogeny (left) constructed with concatenated 16S rRNA, CO1 and 28S rRNA genes, and SMC phylogeny (right) with 16S rRNA. Nodes with bootstrap values lower than 50% were collapsed. Colors are as in Fig 3.

Fig 5. Cophylogenetic reconstruction of scorpion hosts and their SMC symbionts obtained with Jane 4.

The individual phylogenies were constructed and inferred as in Fig 3. Black branches delineate the scorpion phylogeny. Blue branches represent SMC phylogeny. The polytomies formed after collapsing nodes with bootstrap values lower than 50% are shown in purple. Red circles outlined at the nodes indicate suggested cospeciation events.