3.1. Consortia and Replacements in the Auchenorrhyncha
A second large group of sap-sucking insects of the order Hemiptera, extensively studied for the presence of P-endosymbionts, is the Auchenorrhyncha (
Figure 1;
Table 2). This suborder is composed of four superfamilies grouped in two main lineages: Cicadoidea (cicadas), Cercopoidea (spittlebugs), Membracoidea (leafhoppers and treehoppers), grouped in the cicadomorph clade, and Fulgoroidea (planthoppers). Xylem-feeding appears to be an ancestral character of all three modern cicadomorph lineages and has been retained in cicadas, spittlebugs, and a few primitive leafhoppers [
10]. Buchner, and specially his student H. J. Müller, extensively studied this group of insects [
82]. Their microscopic survey of hundreds of species allowed for the observation that most of them contain more than one symbiont, although they all shared a common one that was called at that time “a-symbiont.” Müller’s hypothesis was that the ancestor of the a-symbiont infected the ancestor of the Auchenorrhyncha before the split of the two main clades. He also proposed that the a-symbiont was joined, and sometimes replaced, by one or more additional symbiont types in different descendant host lineages, resulting in the current variety of associations. These hypotheses still hold based on metagenomic studies. This is now considered a perfect example of how the establishment of a symbiotic bacterial consortium can be at the origin of great evolutionary changes in the host’s lifestyle, while the genome degeneration of the consortium partners may end in the extinction and replacement of the more deteriorated and inefficient one, which is similar to what has also been described in aphids.
Müller’s a-symbiont was characterized by phylogenetic and FISH analyses in 2005 as a Bacteroidetes [
83] and was given the name of
Sulcia muelleri, the species name in his honor and the genus
Sulcia after the pioneer symbiologist Karel Šulc. Its phylogenetic congruence with that of the corresponding hosts indicates that modern
Sulcia are descendants of an ancient symbiont that was acquired by the ancestor of all Auchenorrhyncha members, at least 260 MYA. Soon after the description of this new endosymbiotic species, akin to what was found in
C. cedri and almost at the same time, the gammaproteobacterium
Baumannia cicadellinicola was discovered as a co-obligate endosymbiont with
Sulcia in the leafhopper
Homalodisca vitripennis [
84]. However, that was just the beginning. Later on, the availability of genomes of many different
Sulcia strains, as well as genomes of their variable partners in different xylem and phloem-feeding clades, revealed an endless story of alliances and replacements in the evolutionary history of
Sulcia and its symbiotic fellows. We present only the tip of the iceberg of these complicated “family matters” (
Figure 1).
At this time, up to 34 complete
Sulcia genomes are available in GenBank, and in some cases their symbiotic partners have also been sequenced (
Table 2). The analyzed
Sulcia genomes have many features in common with what was found in
Buchnera. An important difference is that, while
Buchnera is the only P-endosymbiont in many aphid lineages,
Sulcia has almost always been detected along with, and complemented by, one or more co-primary microorganisms. The
Sulcia genomes are collinear [
87,
90], and the differences in their gene content imply a perfect metabolic complementation with the additional co-existing endosymbionts to provide their host with essential biomolecules lacking in their nutritionally deficient diet. Very often this involves the partial implementation of a given pathway in each of the partners. The ancestral
Sulcia had an already streamlined genome, as deduced from the very small sizes of the extant
Sulcia genomes that have been sequenced (from 157 to 285 kb).
Three betaproteobacterial species have been identified co-occurring with
Sulcia:
Nasuia deltocephalinicola in phloem-feeding leafhoppers of subfamily Deltocephalinae (family Cicadellidae) from whom it received the species name [
98] but also of family Membracidae [
96],
Zinderia insecticola in many spittlebugs (Cercopoidea) [
90], and
Vidania fulgoroideae in planthoppers (Fulgoroidea) [
99].
Zinderia and
Nasuia (collectively named
BetaSymb clade) are very closely related [
94], which indicates that a common ancestor infected the lineage leading to Cicadomorpha early after the establishment of the endosymbiosis with
Sulcia.
Vidania appears to be a descendant of the ancient symbiont that infected the common ancestor of superfamily Fulgoroidea at least 130 MYA [
82]. The genome reduction syndrome has been dramatic and these symbiotic relationships lead to some of the most highly reduced genomes sequenced to date (down to 112 kb for
Nasuia ALF and PUNC, found in two leafhoppers of genus
Macrosteles) [
94,
95]. These tiny genomes are below the minimal genome status because they have lost genes needed for the maintenance of a living cell (including DNA replication, transcription, and translation) [
69,
100]; therefore, even these essential functions must be performed in cooperation and shared by the joined symbiotic partners.
While
Sulcia—like
Buchnera—is rarely lost, the Beta-endosymbionts have been secondarily lost many times and, akin to what has been described in Lachninae aphids, in most cases they have been replaced by different “healthier” partners [
8]; in other cases, a third partner joined the consortium to cope with the extreme genome degeneration of the two oldest co-primary endosymbionts (
Figure 1). Thus, in the
BetaSymb clade,
Zinderia has been replaced by the alphaproteobacterium
Hodgkinia cicadicola in cicadas (Cicadoidea) [
94], and
Nasuia has been replaced by
Baumannia in subfamily Cicadellinae [
84,
91,
92,
101] and, although it has been retained in most analyzed members of the sister subfamily Deltocephalinae, it seems to have been lost in the corn leafhopper,
Dalbulus maidis [
93,
102]. In the Fulgoroidea,
Vidania and
Sulcia have been found together with the gammaproteobacterium
Purcelliella pentastirinorum in several planthoppers of family Cixiidae [
82,
97,
99,
103].
In many cases, the new allied endosymbiont has been recruited from the same clades that have been repeatedly found as pathogens or facultative symbionts in other sap-feeding insects, suggesting that they can be an environmental source for symbiont exchange and evolutionary reinvigoration when the P-endosymbiont cannot cope with its symbiotic function. As mentioned before, in aphids there are cases in which intracellular symbiotic yeasts have replaced
Buchnera. Similarly, there are many cases in which YLS phylogenetically related to the entomopathogenic genus
Ophiocordyceps (Ascomycota: Sordariomycetes: Hypocreales) have joined or replaced
Sulcia and/or its bacterial partner. Thus,
Sulcia has been replaced in some young lineages of Delphacidae planthoppers (Fulgoroidea) [
107]; neither
Vidania nor
Sulcia were found in some young lineages of family Delphacidae where a vertically transmitted YLS was found [
107,
108]; and the same kind of YLS was found in some leafhoppers of subfamilies Deltocephalinae [
109,
110,
111] and Ledrinae [
112], whether replacing
Nasuia or in an intermediate stage in which both
Nasuia and the YLS coexist with
Sulcia. In addition, many independent cases of
Hodgkinia replacement have been found in several tribes of two cicadidae subfamilies; in fact, it is possible that repeated
Hodgkinia-fungus and fungus–fungus replacements had occurred [
85]. Located in the fat body of the insects, these YLS play an essential role in uric acid recycling [
107,
113]. It is worth mentioning that, in the cases of
Buchnera replacement in aphids, the YLS serves a different endosymbiotic purpose [
77]. As for common environmental and facultative symbiotic bacteria, several
Sodalis-like symbionts have participated in replacements or tripartite associations in the Auchenorrhyncha. For example, in superfamily Cercopoidea, a replacement of
Zinderia by a
Sodalis-like symbiont or intermediate tripartite consortia have been described in spittlebugs of the tribe Philaenini [
89,
114]. In superfamily Fulgoroidea, a
Sodalis-like symbiont has joined the association between
Sulcia and
Vidania in
Caliscelis bonelli (Caliscelidae) [
115]. In all the above-mentioned associations, each endosymbiont lives in its own bacteriocytes, and, in some cases, additional putative facultative symbionts have been also found [
107,
116,
117,
118].
Among all the diversity of endosymbiotic systems found in Auchenorrhyncha, the most widely studied correspond to cicadas, since it is in this superfamily that the most bizarre endosymbiont genomes have been found. For this reason, they deserve their own subsection in this review.
3.2. The Peculiarities of the Hodgkinia Genomes and Its Coexisting Interdependent Lineages within a Single Host
The genomes of different
Hodgkinia strains have been sequenced, and every new genome analyzed provided new surprises. The first genome, from the P-endosymbiont of the glassy-winged sharpshooter
Diceroprocta semicincta [
119], had a GC base composition of 58.4%, very different from all other endosymbiont genomes sequenced at that time. Most strikingly, the genome annotation revealed that the UGA stop codon was reassigned to tryptophan in this bacterium. The same codon reassignment was later detected in the tiny genomes of
Zinderia and
Nasuia [
90,
94], even though they were AT-rich, ruling out the hypothesis that base composition is a root cause of codon reassignment. It is worth mentioning that the same genetic-code modification had been previously described in the reduced and AT-rich genomes of mycoplasmas [
120] and some mitochondrial lineages [
121], representing a remarkable example of evolutionary convergence. It has been proposed that this reassignment was triggered by the loss of release factor RF2 (encoded by
prfB), whose function is to recognize this stop codon [
119].
Nevertheless, what makes
Hodgkinia strains extraordinary is their capability to present alternative interdependent lineages, with different genotypes and genome rearrangements, inside a given host. The coexistence of two cytologically distinct but metabolically interdependent
Hodgkinia clades, with reciprocal patterns of gene loss and retention, was first detected in some cicadas of genus
Tettigades [
88]. Soon later, an impressive level of genome complexity was described in the longest-lived cicadas of genus
Magicicada [
86,
122]. In each
Magicicada species, the
Hodgkinia genome is composed by many subgenomic circles of different size, with an extremely reduced gene density. Additionally, the same gene can be present in different circles, and not all circles are present in all
Hogkinia cells within a single host. Similarly to other highly reduced endosymbiont genomes, and contrary to the first
Hodgkinia genomes sequenced, most of these complex
Hodgkinia genomes have a low GC content (e.g., among the 39 sequenced subgenomic circles of the
Hodgkinia found in
M. neotredecim, the GC content ranges from 21.9 to 42.4, with only three circles having a GC content above 35% [
122]), an indication that a high GC content is not a general trait of this species. Remarkably, genome instability and expansion because of the increase of “junk DNA,” leading to the existence of subgenomic molecules with low coding capacity, are common in mitochondrial lineages from some plants, another example of evolutionary convergence. More recently, an astonishing level of complexity was detected when 19 different
Hodgkinia genomes, isolated from only five specimens from diverse Chilean populations of
Tettigades spp., were sequenced [
87]. The results suggest that a single ancestral
Hodgkinia lineage has split at least six independent times in this insect genus over the last 4 million years. Two to six
Hodgkinia lineages can be found in each single host, and each lineage presents different genomes formed by subgenomic molecules. Each lineage genome contains a different set of genes and, most of the time, does not contain all genes needed for the symbiotic relationship nor the provision of essential informational bacterial functions (i.e., DNA replication, transcription, and translation). Therefore, these different lineages coexisting in a single host rely on each other to survive. Furthermore, different combinations of
Hodgkinia lineages can be found in each host. The degenerative process leading to this amazing splitting phenomenon is progressive and has no way back, since it will be impossible to recover the genomic information that has been lost. The extreme degeneration of these genomes appears to indicate that this endosymbiont has reached a critical stage in genome erosion and could be close to collapse, which could be the cause of the large number and variety of replacements found in this superfamily of insects.