# Sperm mtDNA Copy Number Is Not Associated With Midpiece Size Among Songbirds

## TL;DR

This study found no significant link between the size of the midpiece in songbird sperm and the number of mitochondrial DNA copies.

## Contribution

The study provides new empirical data on mtDNA copy number variation in wild songbird sperm across diverse species.

## Key findings

- Songbird species differ in average mtDNA copy number, but the variation is small.
- There is no significant relationship between midpiece length and mtDNA copy number.
- Mitochondrial genome elimination during spermatogenesis may explain the low variation.

## Abstract

Tremendous variation in sperm morphology is observed across the animal kingdom. Within avian taxa, the songbirds (infraorder Passerides) have the largest variation in sperm morphology. Their spermatozoa move by using energy generated in the midpiece, which is formed by multiple mitochondria fusing together during spermatogenesis. However, very little is known regarding the number of mitochondria required to form the songbird midpiece. Based on previous research showing an association of midpiece length and mitochondrial DNA (mtDNA) copy number in the zebra finch
Taeniopygia guttata
, we hypothesize that songbird species with longer sperm midpieces have more copies of mtDNA. We estimated the sperm mtDNA copy number in 19 species from 10 families within Passerides, covering a broad range of midpiece sizes. Mitochondrial and nuclear DNA abundance were determined using droplet digital PCR (ddPCR) and the ratio between mitochondrial and single‐copy nuclear genes was used to estimate mtDNA copy number per spermatozoon. We found that species differ in their average mtDNA copy number, but the variation was small and not significantly related to midpiece length. A possible explanation is that mitochondrial genomes are eliminated in the spermatids during spermatogenesis.

We investigated mtDNA copy number in 19 wild passerine species representing more than a 100‐fold variation in midpiece length. Using highly accurate ddPCR quantification technology, we show that wild songbird spermatozoa have a relatively low interspecific variation of mtDNA content.