Integrative taxonomy for the traditional coccidians (Chromista: Miozoa: Eimeriidae) from island canaries (Aves: Passeriformes: Fringillidae): Worldwide distribution, morphological and molecular characterization, revaluations and establishment of junior synonyms

Island canaries Serinus canaria (Linnaeus) are finches native to the North Atlantic Islands, however, they have a worldwide distribution in captivity due to their relevance as a pet bird. Coccidians are the most reported parasites of passerines worldwide, both in the wild and in captivity, being frequently associated with disease in passerines kept in rehabilitation centers and commercial breeders. This study aimed to identify coccidians from island canaries kept in captivity in Brazil. Three hundred and fifteen genomic DNA extracted from fecal samples of island canaries from different breeders from Southern and Southeastern Brazil were used to perform a nested PCR assay to amplify a partial fragment of the 28S small subunit ribosomal RNA gene (28S) of Isospora spp. Microscopic screening and morphological identification of Isospora oocysts was performed in fecal samples corresponding to PCR positive DNA samples. Fecal samples have been formalin-stored for approximately four years. Positivity rate for both microscopy and PCR was 10.5% (33/315). Posteriorly, Isospora serini (Aragão, 1933) Box, 1975 and Isospora canaria Box, 1975 were morphologically identified from fresh fecal samples of island canaries maintained by a breeder in the State of São Paulo, Southeastern Brazil, providing a genotypic characterization via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (COI) and 28S genes. The 28S and COI sequences referring to the morphological identification of I. canaria was, respectively, 100% and 99% similar to sequences deposited as Isospora serinuse Yang, Brice, Elliot & Ryan, 2015 from island canaries kept in a rehabilitation center in Australia. The COI sequence referring to the morphological identification of I. serini was 100% similar to a sequence of an extraintestinal Isospora, corroborating this identification/sequencing since I. serini is the first isosporan with an extra-intestinal cycle demonstrated. The comparison of morphological and molecular data from I. canaria and I. serini from this study with published data of Isospora spp. from canaries worldwide, allowed the specific identification from preliminary generic identifications, correction of misidentifications, as well as the establishment of junior synonyms. Finally, this study provides morphological and molecular data that ensure the correct identification of the two Isospora spp. from island canaries in future studies worldwide.

Abstract Island canaries Serinus canaria (Linnaeus) are finches native to the North Atlantic Islands, however, they have a worldwide distribution in captivity due to their relevance as a pet bird. Coccidians are the most reported parasites of passerines worldwide, both in the wild and in captivity, being frequently associated with disease in passerines kept in rehabilitation centers and commercial breeders. This study aimed to identify coccidians from island canaries kept in captivity in Brazil. Three hundred and fifteen genomic DNA extracted from fecal samples of island canaries from different breeders from Southern and Southeastern Brazil were used to perform a nested PCR assay to amplify a partial fragment of the 28S small subunit ribosomal RNA gene (28S) of Isospora spp. Microscopic screening and morphological identification of Isospora oocysts was performed in fecal samples corresponding to PCR positive DNA samples. Fecal samples have been formalin-stored for approximately four years. Positivity rate for both microscopy and PCR was 10.5% (33/315). Posteriorly, Isospora serini (Aragão, 1933) Box, 1975 and Isospora canaria Box, 1975 were morphologically identified from fresh fecal samples of island canaries maintained by a breeder in the State of São Paulo, Southeastern Brazil, providing a genotypic characterization via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (COI) and 28S genes. The 28S and COI sequences referring to the morphological identification of I. canaria was, respectively, 100% and 99% similar to sequences deposited as Isospora serinuse Yang, Brice, Elliot & Ryan, 2015 from island canaries kept in a rehabilitation center in Australia. The COI sequence referring to the morphological identification of I. serini was 100% similar to a sequence of an extraintestinal Isospora, corroborating this identification/sequencing since I. serini is the first isosporan with an extra-intestinal cycle demonstrated. The comparison of morphological and molecular data from I. canaria and I. serini from this study with published data of Isospora spp. from canaries worldwide, allowed the specific identification from preliminary generic identifications, correction of misidentifications, as well as the establishment of junior synonyms. Finally, this study provides morphological and molecular data that ensure the correct identification of the two Isospora spp. from island canaries in future studies worldwide.

Introduction
The island canary Serinus canaria (Linnaeus) is a passerine finch of the Fringillidae family native to the North Atlantic Islands, mainly to the Azores, Madeira, and Canary Islands; however, this species has a worldwide distribution in captivity as pet bird (Dietzen et al., 2006) (Fig. 1). Despite the relevance of S. canaria as a pet bird and the high prevalence of isosporosis in passerines kept in captivity (Page & Haddad, 1995;Dorrestein, 2003;2009), there are few studies related to the diversity and distribution of coccidians from S. canaria. Infection by coccidians of Isospora genus is the major parasitic disease of Passeriformes (Page & Haddad, 1995;Dorrestein, 2003;2009). Acute isosporosis in Passeriformes results in poor digestion, lower nutrients absorption, and weight loss, in addition to high mortality in severe infections (Parenti et al., 1986;Giacomo et al., 1997;Quiroga et al., 2000;Hõrak et al., 2004;Sánchez-Cordón et al., 2007;Maslin & Latimer, 2009;Papini et al., 2012;Oliveira et al., 2018;Gosbell et al., 2020).
Four Isospora spp. have been described or reported from S. canaria: Isospora canaria Box, 1975 and Isospora serini (Aragão, 1933) Box, 1975 are the traditional species of S. canaria, deeply studied by Edith D. Box in the 70's and 80's, since the original observation of the endogenous phases of I. serini by Dr. Henrique B. Aragão in Brazil in the 1930s (Aragão, 1933;Box, 1966;1967;1970;1975;1977;1981;Speer & Duszynski, 1975;Novilla & Box, 1986) (Fig. 1). Isospora canaria was characterized with asexual and sexual cycles limited to intestinal epithelial cells; while I. serini has partial extra-intestinal asexual cycle in organs such as liver, lungs, and spleen, and partial asexual cycle and sexual cycle in the intestinal epithelium (Box, 1975;1977;1981). Information related to molecular sequences of I. canaria and I. serini have yet not been made available. Yang et al. (2015)  kept in a rehabilitation center in Australia, with morphological and molecular data. More recently, Isospora bioccai Cringoli & Quesada, 1991, which was originally described from Oriental greenfinches Chloris sinica (Linnaeus) in Italy (Cringoli & Quesada, 1991), was reported from four canaries in Mexico from morphological data (Luna-Castrejón et al., 2018). In Brazil, Freitas et al. (2003) observed Isospora spp. from 167/327 (50.5%) fecal samples of S. canaria from a breeder in Northeastern Brazil. From a breeder in the State of Rio de Janeiro, Berto et al. (2013) identified I. canaria from 15 S. canaria through morphological data (Fig. 1).
Thus, owing to the lack of information on the specific distribution of Isospora spp. from S. canaria and, mainly, due to the lack of molecular data from sequencing of the traditional coccidian species from S. canaria for comparison with the molecular and morphological data of the recently described/reported species, the aim of this study was to perform the detection and morphological and molecular characterization of Isospora spp. from fecal samples of island canaries S. canaria bred in two States of the Southern and Southeastern Brazil.

Sample collection
Primarily, samples from genomic DNA were obtained from 315 fecal samples from asymptomatic island canaries kept in 63 captivities in Southern (Paraná) and Southeastern (São Paulo) Brazilian states ( Fig. 1) and exhibited at the 64th Ornithological Championship 2015 of the Ornithological Federation of Brazil (FOB), from 07/09/2015 to 07/19/2015, in the Municipality of Itatiba, State of São Paulo, Brazil (Camargo et al., 2018). Fecal samples were collected in the morning and contained feces from the afternoon and night periods from the previous day, taking into consideration the circadian variation in oocyst shedding of Isospora spp. in Passeriformes (López et al., 2007;Dolnik et al., 2011;Coelho et al., 2016). These samples were submitted to Sheather centrifugal-flotation technique and divided in two aliquots, which were processed for genomic DNA extraction and stored at -208 C or preserved in 10% formalin at 48 C. Both aliquots were stored for approximately four years. Furthermore, molecular, morphological, and morphometrical studies were performed for isosporan oocysts recovered from fresh fecal samples of island canaries kept in captivity in the Municipality of Araçatuba, State of São Paulo, Brazil, and collected in October 2020 and May 2022. Fecal samples were submitted to Sheather centrifugal-flotation technique followed by DNA extraction using the Quick DNA Fecal/Soil Microbe Miniprep Kit (Zymo Research), following the manufacturer's instructions.

Molecular analyses
PCR amplification of a fragment of the 28S subunit of the rRNA gene by a nested PCR protocol was performed in all DNA samples using the PCR primers 5' TACCCGCTGAACTTAAGC 3' and 5' CMAC-CAAGATCTGCACTAG 3' (*1495 bp) (Mugridge et al., 2000;Schrenzel et al., 2005) and the nested PCR primers 5' CGCGTTCATTGGGATTTG 3' and 5' CCAGCTATCCTGAGAGAAAC 3' (*793 bp). Nested PCR primers were designed in this study using Primer-Blast (https://www.ncbi.nlm.nih.gov/tools/ primer-blast/). Amplification reactions were performed under the following conditions: 22.5 ll of Platinum TM PCR Supermix (Thermo Fisher Scientific), 2.5 mM MgCl2, 200 nM of each primer and 2.5 ll of target DNA. PCR protocol was performed using DNA denaturation at 948 C for 2 min, followed by 40 cycles, each consisting of denaturation at 948 C for 30 s, 30 s of annealing at 508 C and 90 s of extension at 728 C, with final extension at 72°C for 2 min. Nested PCR protocol was performed under the same PCR conditions, except annealing at 528 C and extension for 1 min.
Further molecular identification was performed in fresh fecal samples recovered in October 2020 and May 2022 by a PCR protocol targeting the COI gene using the primers COIKM204 5 GTTTGGTTCAGGTGTTGGTTG 3 and COIKM205 5 ATCCAATAACCGCACCAAGAG 3, which was developed originally by Schwarz et al. (2009) to amplify a fragment of ± 810 bp of Eimeria spp. PCR reactions was performed in a volume of 22.5 ll containing Platinum TM PCR Supermix (Thermo Fisher Scientific), 2 mM MgCl2, 200 nM of each primer and 2.5 ll of target DNA. Samples were denatured at 948 C for 2 min, followed by 40 cycles, each consisting of denaturation at 948 C for 30 s, 30 s of annealing at 608 C and 1 min of extension at 728 C, with final extension at 72°C for 2 min.
For all PCR protocols, genomic DNA from Isospora spp. isolated from fecal samples of chestnut-bellied seed-finches Sporophila angolensis (Linnaeus) and ultrapure water were used as positive and negative controls, respectively. Amplified fragments were analyzed by GelRed TM (Biotium) stained gel electrophoresis. Nested PCR amplicons submitted to genetic sequencing were purified using the Illustra TM ExoProStar 1-Step purification kit (GE Healthcare Life Sciences).
Molecular cloning was performed in PCR amplicons from COI and 28S rRNA PCRs performed in samples collected in May 2022. PCR amplicons were purified using the PureLink TM Quick Gel Extraction Kit (Thermo Fisher Scientific) and cloned using the TransformAid TM Bacterial Transformation Kit (Thermo Fisher Scientific) and the CloneJET TM PCR Cloning Kit (Thermo Fisher Scientific). Plasmid DNA was purified with the GenElute TM HP Five-Minute Plasmid Miniprep Kit (Sigma-Aldrich).
Genetic sequencing of plasmid DNA and PCR amplicons were accomplished at the Sequencing and Functional Genomics Center of UNESP, Campus of Jaboticabal, using the ABI Prism TM Dye Terminator 3.1. Sequencing was performed in both directions using the nested PCR primers from 28S rRNA and PCR primers from COI PCR protocols. Consensus sequences were determined using CodonCode Aligner version 9.0.1 (CodonCode Corporation) and aligned with homologous sequences using Clustal W ( Thompson et al., 1997) and the Bioedit Sequence Alignment Editor (Hall, 1999).
Phylogenetic analyses were conducted in MEGA X (Kumar et al., 2018) using maximum likelihood analysis based on the Tamura-Nei model (Tamura & Nei, 1993) and the general time reversible model (Nei and Kumar 2000) for the 28S rRNA and COI genes, respectively. Substitution models and optional parameter sets were chosen using the model selection option in MEGA X. The bootstrap consensus trees were inferred from 1000 replicates (Felsenstein, 1985).
Nucleotide sequences generated in this study were submitted to the GenBank database under the accession numbers MW854026, MW854027, OP186067, and OP186068.

Morphological analyses
PCR positive samples, both in formalin and fresh samples, were examined by microscopy for detection and morphological and morphometrical studies of the oocysts, using the technique described by Duszynski & Wilber (1997) and Berto et al. (2014). Microscopic observations, line drawings, photomicrographs and measurements were made using an Olympus BX binocular microscope (Olympus Optical, Tokyo, Japan) coupled to a digital camera Eurekam 5.0 (BEL Photonics, Monza, Italy). Line drawings were edited using Corel DRAW TM and Corel PHOTO-PAINT (Corel Draw Graphics Suite, Version 2020, Corel Corporation, Canada). All measurements are in micrometers and are given as the range followed by the mean in parentheses. The classification of parasites and hosts followed Ruggiero et al. (2015) and Pacheco et al. (2021).

Prevalence and identification
Nested PCR identified 33/315 (10.5%; CI: 7.6-14.3) samples positive for Isospora spp. (Table 1) from the island canaries kept in captivities in Southern and Southeastern Brazil. All samples positive by nested PCR were positive by microscopy.
Due to the storage period of more than four years in 10% formalin, the specific morphological identification was not sufficiently reliable, although one morphotype had oocysts predominantly similar to the morphology described for I. serini (Box, 1975). Isosporan oocysts recovered from fresh fecal samples of the island canaries of Araçatuba in October 2020 had morphological and morphometric characteristics similar to oocysts recovered in most formalin preserved samples. From the oocysts recovered from the samples collected in May 2022 two morphotypes were clearly observed: the first morphotype was identified as the extraintestinal species I. serini. The taxonomic summary for this identification/report follows:
As shown in Table 2, the oocysts identified as I. serini and I. canaria in this study were consistent with the original description, with the exception of some unobserved or not highlighted details by Box (1975). Amplicons from the 28S rRNA PCR performed in samples collected in October 2020 showed sequences with 100% genetic similarity with the sequences from the new genotype identified in Southern and Southeastern Brazil. COI sequence from these samples were 100% similar to a sequence (KP658103) from an extraintestinal Isospora sp. (Ogedengbe et al., 2016).
Samples collected in May 2022 had two distinct 28S rRNA clone sequences. One sequence presented 100% genetic similarity to I. serinuse (Yang et al., 2015), and the other sequence had 100% genetic similarity to the new genotype identified in the other samples. Furthermore, a unique sequence from three COI clones from these samples shared 99.1 to 99.3% genetic similarity to sequences KX276860 and KR477879 of I. serinuse, respectively (Yang et al., 2015;2017). This sequence clustered in a separate clade with I. serinuse sequences with high bootstrap support. COI gene sequences from I. serini grouped in a separate clade with an extraintestinal Isospora sp. from S. canaria (KP658103), also with high bootstrap support (Fig. 5) (Ogedengbe et al. 2016).

Discussion
There are no published data on the prevalence of isosporosis in S. canaria. However, one study of Isospora infection using samples from one aviary in Brazil reported positivity of 50.5% (167/327) (Freitas et al., 2003). The low prevalence reported in this study is probably related to the fact that the birds originated from a championship, so it is assumed that the owners have exhibited birds often treated for parasites and with better physical status; therefore, reducing the chance of clinical or subclinical infections and Isospora oocysts shedding.  Box, 1975Box (1975  The morphology and life cycles of the traditional species I. serini and I. canaria were well detailed and evidenced respectively as extra-intestinal and strictly intestinal (Box, 1966;1967;1970;1975;1977;1981). However, recent descriptions and reports of Isospora spp. from S. canaria were superficial and/or insufficient in the morphological study, leading to identifications of new species, dubious identifications, or nonspecific reports such as Isospora sp.
The species described by Yang et al. (2015) as I. serinuse has wide morphological and morphometric compatibility with I. canaria (Table 2). Yang et al. (2015) has great merit for being a pioneer in the molecular identification of an isosporan from S. canaria, but possibly the genetic sequences presented may refer to the species I. canaria previously described morphologically by Box (1975). In the present study, I. canaria identification was performed by morphological and morphometrical analyses of fresh oocysts.
The report of I. bioccai from S. canaria by Luna-Castrejón et al. (2018) is possible and valid because of the recognized host specificity at the host family level (Duszynski & Wilber, 1997;Berto et al., 2011); however, it is unlikely by the fact that I. bioccai was originally described in Italy from oriental greenfinches Chloris sinica (Linnaeus), while this report in S. canaria occurred in Mexico. It is possible that the main characteristic that led to the identification of I. bioccai was the presence of 4 to 10 elongate polar granules, which are also present in smaller numbers in I. serini by the original description by Box (1975). The size of these oocysts observed by Luna-Castrejón et al. (2018) were reasonably larger than those of I. serini, but in the rest of the characteristic features they were fully compatible (Table 2). In this sense, I. serini would have been a safer and more reliable identification of the oocysts reported on by Luna-Castrejon et al. (2018) because of the great and well known polymorphism of coccidian oocysts caused by numerous factors, mainly in captive birds (Fayer, 1980;Berto & Lopes, 2020).
The identification of I. serini in this study was based on the morphologic data studied in the oocysts that were predominantly observed in fresh fecal samples of S. canaria from the city of Araçatuba. These oocysts were fully compatible with the morphological and morphometric description according to Box (1975) ( Table 2). In addition, the COI gene sequences from knob-like to half-moonshaped, 0.7-1.4 9 2.0-3.1 (1.1 9 2.6) prominent, rounded, 1.8-3.0 9 3.0-4.0 (2.3 9 3.5) subspheroidal compact body of membranebound granules, 5.1-7.3 9 4.6-6.9 (6.2 9 5.8) 1 Originally identified as Isospora bioccai Cringoli & Quesada, 1991 2 Originally identified as Isospora serinuse Yang, Brice, Elliot & Ryan, 2015 these fresh fecal samples of S. canaria from the city of Araçatuba presented 100% genetic similarity to a sequence of Isospora sp. (KP658103) from the liver, spleen, lungs, and intestine of a S. canaria presenting systemic isosporosis (Ogedengbe et al., 2016). In this sense, as I. serini is the only species described from S. canaria as having an extraintestinal cycle, the identification of this morphotype/genotype as I. serini becomes coherent. Phylogenetic analysis of the 28S rRNA sequences (Fig. 6) demonstrated that the samples identified as I. canaria are located in the same clade as the I. serinuse isolate described in Australia. The 28S rRNA sequence obtained for I. serini from both formalin and fresh samples, which were 100% identical to each other, sat in a clade with several sequences of extraintestinal Isospora spp. from passerines of different families (Schrenzel et al., 2005). The two sequences of Isospora spp. of S. canaria in Schrenzel et al. (2005) were different by more than 3% with the sequence of I. serini from this study. This result suggests that other extraintestinal Isospora spp. parasitize S. canaria, or that I. serini has high intraspecific genetic differences, as in T. gondii for this same genic region of 28S rRNA (Gagnon et al., 1996;Rahumatullah et al., 2015).
COI sequences KX276860 and KR477879 of I. serinuse have 99.9% genetic similarity and one nucleotide substitution in the 676 pb partial fragment used for phylogenetic analyses in this study (Yang et al., 2015;2017). COI sequences from I. canaria from this study (OP186067) showed 99.1 to 99.3 genetic similarity with sequences KX276860 (six nucleotide substitutions) and KR477879 (five nucleotide substitutions) of I. serinuse, respectively (Yang et al., 2015;2017). Furthermore, genetic similarity between two sequences from Isospora maroninae (KT224377 and KX276861) is 99.6% (three nucleotide substitutions) (Yang et al., 2015;; and the genetic similarities among I. canaria and I. maroninae (KT224377) and Isospora sp. from Parus major Linnaeus, 1758 (MK573833) at the COI gene were 95.5% and 97.2%, respectively (Yang et al., 2016;Trefancová & Kvičerová, 2019). There are insufficient COI sequences of Isospora species from birds published in genetic sequences databases to determine inter-and intraspecific genetic difference in Isospora spp. It is also noteworthy that I. serinuse was identified in Australia and I. canaria was identified in South America in this study, therefore some genotypic variation due to geographic distance can be expected, even for a passerine with a worldwide distribution as a pet bird (Berto & Lopes, 2020). Kubisk et al. (2022) proposed that since that delineation is not clear for COI gene, a combined genetic, morphologic, biologic, and ultrastructural analyses should be performed to identify Isospora species; in other words, an integrative taxonomy. In this sense, the genetic similarity of 99.1 to 99.3 and 100% among COI and 28S rRNA sequences from I. canaria from this study and I. serinuse sequences, respectively; the grouping of I. canaria sequences in the same clade of I. serinuse with high bootstrap support in both 28S rRNA and COI phylogenetic trees, Fig. 6. Phylogenetic consensus tree of the 28S rRNA gene sequences from Isospora serini and Isospora canaria from this study (circles) and selected Isospora spp. according to the maximum likelihood analysis based on Tamura-Nei model. Numbers on the left of the supported nodes indicate the bootstrap values (1000 replicates). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. A discrete gamma distribution was used to model evolutionary rate differences among sites (5 categories (?G, parameter = 0.3995)). The tree was rooted with the sequences of Toxoplasma gondii (L25635) and Neospora caninum (AF001946). This analysis involved 27 nucleotide sequences. There were 732 positions in the final dataset. Evolutionary analyses were conducted in MEGA X. along with morphological and morphometrical analyses of oocysts (Table 2) allowed the establishment of I. serinuse as a junior synonym of I. canaria.

Conclusions
Low prevalence of Isospora spp. were found in the captivities of island canaries S. canaria in Southern and Southeastern Brazil. Isospora canaria and I. serini were morphologically and molecularly characterized for the COI and 28S genes, providing data that ensure the correct identification of these isosporans in future studies worldwide. The report of Isospora sp. by Ogedengbe et al. (2016) and I. bioccai by Luna-Castrejón et al. (2018) were reevaluated and corrected to I. serini and I. serinuse of Yang et al. (2015) was established as a Junior Synonym of I. canaria.
Author contributions The study was designed by BPB and MVM. Samples collection was performed by AAN and ELM. Laboratory procedures for maintenance, recovery, measurements, photomicrographs and isolation of oocysts were performed by ELM and AAN. DNA extraction, PCR amplification, molecular cloning and sequencing were performed by AAN, ELM, CMSH, BMSB and DBRS. BPB drew the coccidian oocysts. The manuscript was written by BPB and MVM and subsequently revised by all other authors. All authors read and approved the final manuscript. Data availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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