Methuselah/Methuselah-like G protein-coupled receptors constitute an ancient metazoan gene family

Inconsistent conclusions have been drawn regarding the phylogenetic age of the Methuselah/Methuselah-like (Mth/Mthl) gene family of G protein-coupled receptors, the founding member of which regulates development and lifespan in Drosophila. Here we report the results from a targeted homolog search of 39 holozoan genomes and phylogenetic analysis of the conserved seven transmembrane domain. Our findings reveal that the Mth/Mthl gene family is ancient, has experienced numerous extinction and expansion events during metazoan evolution, and acquired the current definition of the Methuselah ectodomain during its exceptional expansion in arthropods. In addition, our findings identify Mthl1, Mthl5, Mthl14, and Mthl15 as the oldest Mth/Mthl gene family paralogs in Drosophila. Future studies of these genes have the potential to define ancestral functions of the Mth/Mthl gene family.

Importantly, the Drosophila repertoire of Mth/Mthl gene family members also includes four paralogs that lack significant similarity to the Mth ectodomain, but were identified based on conserved sequence signatures in the 7TM domain: Mthl1, Mthl5, Mthl14, and Mthl15 9,16 . The 7TM domain has thus been recognized as a more conserved sequence region of the Drosophila Mth/Mthl gene family, which includes both Mth ectodomain-positive and -negative members.
The significance of the 7TM domain is further underlined by the fact that some 7TM domain-based studies reported evidence of candidate Mth/Mthl homologs in metazoan species outside arthropods: the sea squirt Ciona intestinalis 18 , the lancelet Branchiostoma floridae, and the sea anemone Nematostella vectensis 5 . At the same time, no candidate Mth/Mthl homologs were noted in the first genome-wide survey of GPCRs in the lancelet 19 or in similar studies of the acorn worm Saccoglossus kowalevskii 20 and flatworms 21 , possibly due to incomplete genome sequence coverage or sampling. Finally, a recent study of GPCR diversity in insects expressed doubt about the monophyly of the Mth/Mthl subfamily 22 .
These confounding data leave three major questions: How deeply conserved is the Mth/Mthl gene family in the metazoan tree of life? Is the Mth ectodomain an ancestral or derived component of Mth/Mthl gene family members? And which are the most ancestral Mth/Mthl homologs in Drosophila that could give insights into the earliest functions of the Mth/Mthl gene family?

Results and Discussion
Early metazoan origin of the Mth/Mthl gene family. To improve our understanding of the Mth/Mthl gene family, we investigated the phylogenetic position of previously and newly identified candidate homologs. To this end, we searched a database of 39 genomes representing holozoan species diversity (Ichthyosporea + C orallochytrium + Filasterea + Choanoflagellatea + Animalia) (Supplementary Table S1) by BlastP with the 7TM domain of D. melanogaster Mthl1 as query and collected the 100 best hits as candidate homologs. We then used the best matching human candidate homolog (GPR98) from this pool as query for a second search, from which we collected an additional 100 best matching sequences after removing duplicates found in the first search. In parallel, we identified candidate homologs from species without complete genomes based on reciprocal BLAST evidence using the previously reported Mthl candidate homolog from N. vectensis as query 5 . Combined, these efforts resulted in a total of 278 GPCR sequences (Supplementary text file S1), which were used to build a multiple alignment of the 7TM domain (Supplementary text file S2) for molecular phylogenetic gene tree estimation using Bayesian and likelihood approaches.
Consistent with previous studies, the resulting trees recovered a robustly supported clade of arthropod Mth/Mthl homologs ( Fig. 1A and Supplementary text files S3 and S4). This clade was nested within a likewise robustly supported, more inclusive clade. Altogether, our trees identified 44 Mth/Mthl candidate homologs from diverse animal species outside insects. These included protostomes (Annelida, Mollusca), invertebrate deuterostomes (Echinodermata, Hemichordata, Cephalochordata), and the sea anemone N. vectensis.
To scrutinize the non-insect candidate homologs, we performed reciprocal BLAST searches against the NCBI nr protein sequences of D. melanogaster and B. floridae. This approach defined 24 high confidence Mth/Mthl homologs outside insects, which included 10 other arthropod, 4 mollusc, 7 hemichordate, and 3 cnidarian genes 5 (Supplementary Table S2). Our targeted, double pronged approach thus corroborated the previously suggested presence of Mth/Mthl homologs in sea squirts 18 , lancelets, and cnidarians 5 and, for the first time, detected Mth/Mthl homologs in the acorn worm S. kowalevskii 20 .

Mth/Mthl gene family extinctions and expansions. Within chordates, our analyses only detected Mth/
Mthl homologs in the lancelet species B. floridae and B. belcheri (Fig. 1). To further explore the apparent absence of the Mth/Mthl gene family in other chordate clades, we searched the NCBI nr database by reciprocal BlastP for tunicate and vertebrate homologs using the B. floridae Mth/Mthl homologs XP_002610765 and XP_002608881 as queries (Fig. 1). Also this approach failed to detect Mth/Mthl homologs in either tunicates and vertebrates, leading to the conclusion that the Mth/Mthl gene family became extinct during early chordate evolution and was absent in the last common ancestor of urochordates and vertebrates.
Further losses of the Mth/Mthl gene family during metazoan evolution were indicated by absence of detectable Mth/Mthl homologs in genomes from Nematoda, Platyhelminthes, and Rotifera. A particularly prominent example in addition to vertebrates was the absence of Mth/Mthl homologs in the cnidarians Acropora digitifera and Hydra magnipapillata in contrast to the presence of three Mth/Mthl homologs in N. vectensis ( Fig. 1 and Supplementary Tables S2 and S3).
Our trees also detected multiple independent expansions of the Mth/Mthl gene family in the metazoan tree of life. The most dramatic example of this continues to be the exceptionally enlarged Mth/Mthl gene family cluster of insects ( Fig. 1)  Derived acquisition of the Mth ectodomain. The Mth ectodomain is defined by 10 cysteine residues, which form a total of five disulfide bonds 7,16 . Most of these residues were noted to be conserved in the Drosophila paralogs Mthl2-4, and Mthl 7-12 but not in Mthl1 and Mthl5 16 . Consistent with this, we were able to confirm the presence of the Mth ectodomain for Mthl2-4, and Mthl 7-12 in Pfam database searches based on significant matches to the Methuselah_N domain PF06652 with e-values between 8.9E-18 to 3.1E-69 but not for Mthl1 and Mthl5 (Supplementary Table S3). Moreover, Pfam support for the presence of a Mth ectodomain was exceptionally low or non-significant for the more recently described Drosophila paralogs Mth15 (Pfam e-value: 0.00012) and Mthl14, respectively.
To  Table S3). Almost all of these represented insects except for the previously reported custacean Mth/Mthl homolog EFX89685 from Daphnia (Pfam e-value: 0.00012) 14 .
Mapping the conservation of the Mth ectodomain onto the 7TM domain-based Mth/Mthl gene family tree further revealed that all Mth ectodomain-positive homologs are contained in the strongly expanded arthropod subcluster of Mth/Mthl homologs (Fig. 1B) and that this clade is sister to the Mth ectodomain-negative homolog Mthl5. Since Mth/Mthl gene family members outside arthropods are all Mth ectodomain-negative, outgroup rooting supports the model that the Mth ectodomain is a derived domain that has been acquired during arthropod evolution.
Interestingly, our Pfam searches detected evidence of additional ectodomain acquisition events in the Mth/Mthl gene family tree. This included the presence Somatomedin_B domains in the Mth/Mthl homologs XP_001628572 of N. vectensis, XP_002608881 of B. floridae, and XP_006821924 of S. kowalevskii (Fig. 1B). The evolution of the Mth/Mthl gene family may thus have been repeatedly shaped by ectodomain acquisition events. 51 Mth/Mthl gene family members in our trees possess ectodomains that lack significant similarities to any currently known protein domains. This includes Mthl1, Mthl5, and Mthl14, which, according to our trees, represent the oldest Mth/Mthl gene family members in Drosophila, together with Mthl 15, which is characterized by only marginal Mth ectodomain support (0.00012). The phylogenetic evidence thus suggests that defining the ligand binding properties and physiological functions for these Drosophila homologs has the potential to elucidate ancestral functions of the Mth/Mthl gene family.

Candidate ancestral Mth/Mthl receptors in Drosophila.
Interestingly, expression studies in Drosophila point toward a role of Mthl5 in the development of the visceral and cardiac mesoderm 9 . Moreover, recent gene knockdown studies in the red flour beetle Tribolium castaneum produced evidence that Mthl5 is essential for embryonic and postembryonic survival 23,24 , interacting with several signaling pathways 25 . In combination, these first insights and the strong gene tree evidence of an ancestral status of Mthl5 prioritize this paralog for further study.

Methods
The multiple sequence alignment was built using MAFFT, applying the L-INS-i method 26 and manually cleared of gaps and non-homologous regions outside the 7TM domain. The trimmed alignment of 365 amino acid positions was subjected to phylogenetic analysis with PhyloBayes 3.0 until 2 chains converged (maxdiff < 0.3) 27 and to estimate the 20 best RaxML 8.0 trees 28 using the LG model and 8 categories for gamma distribution and 100 bootstrap replicates. Pfam searches were performed at default gathering threshold 29 . Species lists, sequences, alignments, and trees are available in Supplementary Information.