CHAPTER 1. THE PILOT RESEARCHES FOR EVOLUTIONARY
2.3. General discussion
2.3.1. The ab initio prediction and annotation of marine arthropod Hox genes
The Hox genes are evolutionarily conserved transcription factors containing homeodomain motifs. They play critical roles in the patternization of the embryonic segments throughout the anterior-posterior axis. The hypothetical ancestral arthropod genome is suggested to contain 10 Hox genes (labial, proboscipedia, Hox3, Deformed, Sex combs reduced, fushi tarazu, Antennapedia, Ultrabithorax, abdominal-A, and Abdominal-B) as a well conserved cluster (Akam et al., 1994; Pace et al., 2016). In addition, these 10 Hox genes are usually found to be organized in a cluster as the same order of their domains of expression throughout the embryonic anterior-posterior axis, which is typically described as spatial collinerarity (Pace et al., 2016). These Hox genes have been intensly studied within diverse clade in the Phylum Arthropoda which supported 10 Hox genes generally conserved (Cook et al., 2001; Hughes and Kaufman, 2002). On the other hand, the conservation of their genomic arrangements remains unclear throughout the phylum, due to the majority of researches focusing on to the subphylum Hexapoda (Pace et al., 2016). Therefore, most of the cases from non-hexapod arthropod Hox gene studies have been conducted as gene-based surveys, and some early studies could not recover several Hox genes from decapod species (Mouchel‐Vielh et al., 1998; Abzhanov and Kaufman, 2000; Deutsch and Mouchel‐Vielh, 2003). Among pycnogonids, the study on their Hox genes also was not able to recover some Hox genes, such as abdominal-A (Manuel et al., 2006).
Nevertheless, considering that few examples of Hox gene losses were reported witin arthropods, the loss of several core Hox genes from decapods and pycnogonids are not plausible (Pace et al., 2016). In recent genomic studies, however, entire 10 Hox genes
were present in de novo assembled decapod shrimp genomes, such as Neocaridina denticulata (Kenny et al., 2014), Penaeus japonicus (Yuan et al., 2018) and P. vannamei (Zhang et al., 2019). In addition, all 10 Hox genes were recovered from the Portunus trituberculatus and Chionoecetes opilio genomes in this study. On the other hand, fushi tarazu was absent in E. carinicauda genome (Yuan et al., 2017) and proboscipedia was not present in P. monodon genome (Yuan et al., 2018), which was unnatural considering that their closely related species show fully recovered 10 Hox genes (Figure 19). Since both of E. carinicauda and P. monodon demonstrate highly uniform “decapod shrimp”
morphology, their lack of fushi tarazu and proboscipedia is possibly resulted from incomplete genome assembly or gene prediction. Except P. vannamei, all referred decapod shrimp genomes were de novo assembled solely from the short read-lengthed Illumina paired-end sequenced reads, thus their genomic assemblies consist with more than a million scaffolds with N50 value less than 1,000 bases long (Table 2).
As previously discussed in Chapter 2.1, these highly fragmented genome assemblies result in the overestimated numbers of fragmented genes by splitting the exons of a single gene into different fragmented genomic scaffolds. Furthermore, Hox genes, whose expressions are highly limited in the embryonic development, are more vulnerable being not detected when the sufficient clues from embryonic transcriptomes or the sequences of homologous genes from closely related species cannot be provided. The presence of all 10 Hox genes from the Portunus trituberculatus and Chionoecetes opilio genomes therefore further validate the quality of their assemblies and also suggest that the decapod ancestor contained all these Hox genes as more ancient, arthropod ancestor did.
In contrast to two brachyuran crab genomes in this study, there were only 9 recognizable Hox genes present from the Nymphon striatum genome. The abdominal-A
gene was lost from its genome, which accords well with preceeding studies on pycnogonid Hox gene expressions (Manuel et al., 2006; Pace et al., 2016). These studies suggested that the parallel cases of abdominal-A lost in barnacles and chelicerates (pycnogonids and mites) correlated with their morphologies of highly reduced abdomen.
In addition, some Hox genes from Chionoecetes opilio and Nymphon striatum showed unique characteristics from their genomic arrangement. In Chionoecetes opilio genome, 5 Hox genes (deformed, sex combs reduced, Antennapedia, Ultrabithorax, and abdominal- A) are located in the minus strand of a single genomic sequence, scaffold5763. However, abdominal-A is translocated between sex combs reduced and Antennapedia, which disturbs the genomic collinearity well known in hexapods. Furthermore, rest of 5 Hox genes are scattered into different scaffolds (labial and proboscipedia: scaffold3496, Hox3:
scaffold18071, and fushi tarazu: scaffold25914), which is uncommon case among arthropod, while these atomized pattern of Hox genes are reported from mollusk species (Albertin et al., 2015; Kwak, 2017).
More surprisingly, Hox genes of Nymphon striatum are also atomized (labial:
scaffold409, proboscipedia: scaffold386, Hox3: scaffold434, deformed: scaffold170, sex combs reduced and fushi tarazu: scaffold379, Antennapedia: scaffold973, Ultrabithorax and Abdominal-B: scaffold229) despite of its far longer genomic scaffolds compared to those of Chionoecetes opilio. Furthermore, putative duplicated Hox genes were detected from both of Chionoecetes opilio and Nymphon striatum genomes. A partially duplicated Ultrabithorax was present between Antennapedia and orthologous Ultrabithorax in Chionoecetes opilio genome. On the other hand, fragments of labial (scaffold98) and proboscipedia (scaffold20) were found in Nymphon striatum genome. Lastly, the sequence of Nymphon striatum Abdominal-B was greatly truncated when it was aligned
with those of other chelicerates. This finding also accords with the previous diagnosis of pycnogonid Abdominal-B (Manuel et al., 2006) which further supports the correlation suggested by them between pycnogonid reduced abdomen and its truncated posterior Hox genes.
Nevertheless, to test the hypothesis on the abdomen reduction of decapods and pycnogonids in the context of Hox genes evolution, further studies are required. Firstly, Hox genes from various taxa with reduced abdomens and their close relatives with elongated abdomens must be compared each other comprehensively. In addition, Architeuthis dux, a cephalopod, whose genome showed its all core Hox genes located on a single genomic scaffold with especially long intervals, which opposed to the previous cases of atomized Hox genes in mollusk genomes (da Fonsca et al., 2020). This further stress the importance of increase of genomic contiguity up to sub-chromosomal level, therefore the genome assemblies of Chionoecetes opilio, Nymphon striatum, and Portunus trituberculatus require improved scaffolding analyses. Lastly, the Hox genes from three arthropod genome assemblies in this study need to be further curated manually.
Since these Hox genes were predicted with the automated Seqping pipeline alone, it is possible to some of them are erroneously predicted. Therefore, manual curations such as comparing predicted transcript structures, transcriptomic clues and these genes must be conducted appropriately. These further studies are currently in progress, their completed results could not be included in this dissertation.
2.3.2. The optimizied workflow of de novo whole-genome researches of marine